Expanded porphyrins: large porphyrin-like tripyrroledimethine-derived macrocycles

ABSTRACT

The present invention involves a novel tripyrrole dimethine-derived &#34;expanded porphyrin&#34; (texaphyrin), the synthesis of such compounds, their analogs or derivatives and their uses. These expanded porphyrin-like macrocycles are efficient chelators of divalent and trivalent metal ions. Metal complexes of these compounds are active as photosensitizers for the generation of singlet oxygen and thus potentially for inactivation or destruction of human immunodeficiency virus (HIV-1), mononuclear or other cells infected with such virus and tumor cells as well. A variety of texaphyrin derivatives have been produced and many more are readily obtainable. Various metal (e.g., transition, main group, and lanthanide) complexes with the texaphyrin and texaphyrin derivatives of the present invention have unusual water solubility and stability which render them particularly useful. These metallotexaphyrin complexes have optical properties making them unique as compared to existing porphyrin-like or other macrocycles. For example, they absorb light strongly in a physiologically important region (i.e. 690-880 nm). These complexes also form long-lived triplet states in high yield and act as efficient photosensitizers for the formation of singlet oxygen. These properties, coupled with their high chemical stability and appreciable solubility in polar media such as water, add to their usefulness.

This application is a continuation application of Ser. No. 08/168,668filed Dec. 16, 1993, now U.S. Pat No. 5,369,101. U.S. Ser. No.08/168,668 was a divisional application of Ser. No. 07/880,131 filed May7, 1992, since issued as U.S. Pat. No. 5,272,142. Ser. No. 07/880,131was a divisional application of Ser. No. 07/539,975 filed Jun. 18, 1990since issued as U.S. Pat. No. 5,162,509. Ser. No. 07/539,975 was adivisional application of Ser. No. 07/320,293 filed Mar. 6, 1989, sinceissued as U.S. Pat. No. 4,935,498. The copending application and thepatents are incorporated by reference herein.

BACKGROUND OF THE INVENTION

The porphyrins and related tetrapyrrole macrocycles are among the mostversatile of tetradentate ligands¹ (see Example 1 for the references inthis paragraph). Attempts to stabilize higher coordination geometries,however, with larger porphyrin-like aromatic macrocycles have met withlittle success.²⁻⁵ Indeed, to date, only the uranyl complex of"superphthalocyanine" has been isolated and characterized structurally,²although several other large porphyrin-like aromatic macrocycles,including the "sapphyrins",³,6 "oxosapphyrins",⁶,7 "platyrins",⁸"pentaphyrin",⁹ and "[26]porphyrin",¹⁰ have been prepared in their metalfree forms.

Although the porphyrins and related tetrapyrrolic compounds remain amongthe most widely studied of all known macrocycles,¹ relatively littleeffort has been devoted to the development of larger conjugated pyrrolecontaining systems²⁻¹² (references cited in this paragraph are shown inExample 2). Large, or "expanded" porphyrin-like systems, however, are ofinterest for several reasons: They could serve as possible aromaticanalogues of the better studied porphyrins²⁻⁸ or serve as potentialbiomimetic models for these or other naturally occurringpyrrole-containing systems.¹³,14 In addition, large pyrrole containingsystems offer exciting possibilities as novel metal bindingmacrocyles.²,9-12,15 For instance, suitably designed systems could actas versatile ligands capable of binding larger metal cations and/orstabilizing higher coordination geometries²,16 than those routinelyaccommodated within the normally tetradentate ca. 2.0 Å radius porphyrincore.¹⁷ The resulting complexes could have important application in thearea of heavy metal chelation therapy, serve as contrast agents formagnetic resonanace imaging (MRI) applications, act as vehicles forradioimmunological labeling work, or serve as new systems for extendingthe range and scope of coordination chemistry.¹⁵,18 in addition, thefree-base (metal-free) and/or diamagnetic metal-containing materialscould serve as useful photosensitizers for photodynamic therapeuticapplications. In recent years a number of potentially pentadentatepolypyrrolic aromatic systems, including the "sapphyrins",³,4"oxosapphyrins",⁵ "smaragdyrins",³,4 platyrins,⁶ and "pentaphyrin"⁷ havebeen prepared and studied as their metal-free forms. For the most part,however, little or no information is available for the correspondingmetallated forms. Indeed, the uranyl complex of "superphthalocyanine"was the only metal-containing pentapyrrolic system which has beenprepared and characterized structurally.² Unfortunately, the"superphthalocyanine" system is apparently not capable of existence ineither its free-base or other metal-containing forms.² Thus, prior tothe present invention, no versatile, structurally characterized,pentadentate aromatic ligands were available,¹¹ although a number ofnonaromatic pyridine-derived pentadentate systems had previously beenreported.¹⁹,20

Gadolinium(III) complexes derived from strongly binding anionic ligands,such as diethylenetriamine pentaacetic acid (DTPA),¹,2,31,4,7,10-tetraazacyclododecane N,N',N,",N'"-tetraacetic acid(DOTA),¹,4,5 and1,10-diaza-4,7,13,16-tetraoxacyclooctadecane-N,N'-diacetic acid(dacda),¹,6 are among the most promising of the paramagnetic contrastcurrently being developed for use in magnetic resonance imaging (MRI)¹(references cited in this paragraph are shown in Example 3). Indeed,[Gd·DTPA]⁻ is now undergoing clinical trials in the United States forpossible use in enhanced tumor detection protocols.¹ Nonetheless, thesynthesis of other gadolinium(III) complexes remains of interest sincesuch systems might have greater kinetic stability, superior relaxivity,or better biodistribution properties than the existing carboxylate-basedcontrast agents. One approach currently being pursued is based on usingwater-soluble porphyrin derivatives, such astetrakis(4-sulfonatophenyl)porphyrin (TPPS).⁷,8,9 Unfortunately, thelarge gadolinium(III) cation cannot be accommodated completely¹⁰ withinthe relatively small prophyrin binding core (r≅2.0 Å¹¹), and, as aconsequence, gadolinium prophyrin complexes are invariablyhydrolytically unstable.⁷,8,12,13 Larger porphyrin-like ligands,however, might offer a means of circumventing this problem.¹⁴⁻²²

Acquired immunodeficiency syndrome (AIDS) and cancer are among the mostserious public health problems facing our nation today. AIDS, firstreported in 1981 as occurring among male homosexuals,¹ is a fatal humandisease which has now reached pandemic proportions (the references inthis and the following four paragraphs are shown in Example 5). Cancer,in spite of some very significant advances in diagnostics and treatmentin recent years, remains the third leading cause of death in thiscountry. Finding better ways to detect, treat, and reduce thetransmission of these disorders are thus research objectives of thehighest importance.

One of the more promising new modalities currently being explored foruse in the control and treatment of tumors is photodynamic therapy(PDT).¹⁻⁵ This technique is based on the use of a photosensitizing dye,which localizes at, or near, the tumor site, and when irradiated in thepresence of oxygen serves to produce cytotoxic materials, such assinglet oxygen (O₂ (¹ Δ_(g))), from otherwise benign precursors (e.g.(O₂ (³ Σ_(g) -)). Much of the current excitement associated with PDTderives from just this property: In marked contrast to current methods(e.g. conventional chemotherapy), in PDT the drugs themselves can (andshould) be completely innocuous until "activated" with light by anattending physician. Thus a level of control and selectivity may beattained which is not otherwise possible.

At present, diamagnetic porphyrins and their derivatives are consideredthe dyes of choice for PDT. It has been known for decades thatporphyrins, such as hematoporphyrin, localize selectively in rapidlygrowing tissues including sarcomas and carcinomas,⁶ although the reasonsfor this selectivity remain recondite. Currently most attention isfocusing on the so-called hematoporphyrin derivative (HPD)²⁻⁵,7-21 anincompletely characterized mixture of monomeric and oligomericporphyrins produced by treating hematoporphyrin dihydrochloride withacetic acid-sulfuric acid followed by dilute base.²²⁻²⁷ Fractions richin the oligomeric species, which are believed to have the besttumor-localizing ability,²³,26 are marketed under the trade namePhotofirin II® (PII) and are currently undergoing phase III clinicaltrials for obstructed endobronchial tumors and superficial bladdertumors. Here, the mechanism of action is thought to be largely, if notentirely, due to the photoproduction of singlet oxygen (O₂ (¹ Δ_(g))),although alternative mechanisms of action, including those involvingsuperoxide anion or hydroxyl and/or porphyrin-based radicals cannot beentirely ruled out.²⁸⁻³³ Promising as HPD is, it and other availablephotosensitizers (e.g., the phthalocyanines and napthaphthalocynines)suffer from serious disadvantages.

While porphyrin derivatives have high triplet yields and long triplelifetimes (and consequently transfer excitation energy efficiently totriplet oxygen),^(3b),3g their absorption in the Q-band region oftenparallels that of heme-containing tissues. Phthalocyanines andnaphthalocyanines absorb in a more convenient spectral range but havesignificantly lower triplet yields;⁴ moreover, they tend to be quitesoluble in polar protic solvents, and are difficult to functionalise.Thus at present the development of more effective photochemotherapeuticagents appears to require the synthesis of compounds which absorb) inthe spectral region where living tissues are relatively transparent(i.e., 700-1000 nm),^(1d) have high triplet quantum yields, and areminimally toxic. The present inventors have recently reported⁵ (seeExample 1) the synthesis of a new class of aromatic porphyrin-likemacrocycles, the tripyrroledimethine-derived "texaphyrins", which absorbstrongly in the tissue-transparent 730-770 nm range. The photophysicalproperties of metallotexaphyrins 1_(c) -7_(c) parallel those of thecorresponding metallaporphyrins and the diamagnetic complexes 1_(c)-4_(c) sensitize the production of ¹ O₂ in high quantum yield. FIG. 19shows the schematic structure, metal complexes and derivatives ofcompounds of the present invention (1_(c) -7_(c)).

Singlet oxygen is also believed to be the critical toxic speciesoperative in experimental photsensitized blood purificationprocedures.³⁴⁻³⁹ This very new application of photodynamic therapy is oftremendous potential importance: It shows promise of providing a safeand effective means of removing enveloped viruses such as HIV-1, herpessimplex (HSV), cytomegalovirus (CMV), various forms ofhepatitis-inducing virus, as well as other opportunistic blood-borneinfections (e.g. bacteria and malaria plasmodium) from transfused wholeblood. Given that AIDS is currently a not effectively treated andusually fatal disease, the benefit of such a blood purificationprocedure would be of inestimable value.

At present, sexual relations and needle-sharing are the dominantmechanisms for the spread of AIDS.¹ An increasing percentage of AIDSinfections, however, are now occurring as a result of bloodtransfusions.¹,40-43 Unfortunately, banked blood components areessential products for the practice of modern medicine and as a resultthis method of transmission is not likely to be precluded by simplechanges in lifestyle. Rather, an absolutely fail-proof means must bedeveloped to insure that all stored blood samples are free of the AIDSvirus (and ideally all other blood-borne pathogens). To a certain extentthis can be accomplished by screening the donors' histories and carryingout serologic tests. At present, however, the serologic tests for HIV-1are insufficient to detect all infected blood samples, in particular,those derived from donors who have contacted the disease but not yetproduced detectable antibodies.⁴²,43 In addition, new mutants of theAIDS virus have been detected; some or all of which may escape detectionby current means.¹ Thus, an antiviral system is needed which removes anyform of HIV-1 from stored blood. This is particularly important since astored blood sample from one infected donor could potentially end upbeing administered to several different patients, in, for instance, thecourse of pediatric care.

SUMMARY OF THE INVENTION

The present invention involves a novel tripyrrole dimethine-derived"expanded porphyrin" (texaphyrin), the synthesis of such compounds,their analogs or derivatives and their uses. These expandedporphyrin-like macrocycles are efficient chelators of divalent andtrivalent metal ions. Metal complexes of these compounds are effectiveas photosensitizers for the generation of singlet oxygen and thuspotentially useful for the inactivation or destruction of tumors as wellas for the prophylactic treatment and removal of human, immunodeficiencyvirus (HIV-1), and other vital contaminants from blood. A variety oftexaphyrin derivatives have been produced and many more are readilyobtainable. Various metal (e.g., lanthanide) complexes with thetexaphyrin and texaphyrin derivatives of the present invention haveunusual water solubility and stability which render them particularlyuseful. These metallotexaphyrin complexes have optical properties makingthem unique as compared to existing porphyrin-like or other macrocycles.For example, they absorb light strongly in a physiologically importantregion (i.e. 690-880 nm). Certain diamagnetic complexes also formlong-lived triplet states in high yield and act as efficientphotosensitizers for the formation of singlet oxygen. These properties,coupled with their high chemical stability and appreciable solubility inpolar media such as water, add to their usefulness.

The present invention relates to a class of compounds related to thebasic compound having the structure: ##STR1## wherein R is H or CH₃.This compound has been prepared as one having the structure: ##STR2##wherein M is H, L is absent and n is 0; or M is Cd, L is pyridine orbenzimidazole and n is 1.

A preferred compound of this invention is a cadmium-texaphyrin complexhaving the structure: ##STR3##

A variety of texaphyrin derivatives and their metal complexes have beenprepared and may be characterized as having the structure: ##STR4##wherein M is H, R is H and n is 0;

M is Cd⁺², R is H and n is 1;

M is Nd⁺³, R is H and n is 2;

M is Sm⁺³, R is H and n is 2;

M is Eu⁺³, R is H and n is 2;

M is Gd⁺³, R is H, and n is 2;

M is Y⁺³, R is H, and n is 2;

M is In⁺³, R is H, and n is 2;

M is Zn⁺², R is H and n is 1;

M is Hg⁺², R is H, and n is 1.

M is H, R is CH₃ and n is 0;

M is Gd⁺³, R is CH₃ and n is 2;

M is Eu⁺³, R is CH₃ and n is 2;

M is Sm⁺³, R is CH₃ and n is 2;

M is Y⁺³, R is CH₃ and n is 2; or

M is In⁺³, R is CH₃ and n is 2.

The present invention also includes a compound which may be described ashaving the structure: ##STR5## wherein M is Zn⁺², R and R' are H and nis 1;

M is Zn, R is H, R' is Cl and n is 1;

M is Cd, R and R' are H and n is 1;

M is Cd, R is H, R' is Cl and n is 1;

M is Mn, R and R' are H, and n is 1;

M is Sm, R and R' are CH₃ and n is 2;

M is Eu, R and R' are CH₃ and n is 2; or

M is Gd, R and R' are CH₃ and n is 2.

In a broader view, the present invention involves a compound having thestructure: ##STR6## wherein R and R' are CH₃ ; R is H and R' is OCH₃ ; Ris H and R' is Cl; R is H and R is COOH or R is H and R' is NO₂ ; andwherein M is a divalent metal ion and n is 1, or M is a trivalent metalion and n is 2.

In another view the texaphyrin and its derivatives and complexes thereofof the present invention have the structure: ##STR7## wherein M is adivalent metal ion and n is 1, or M is a trivalent metal ion and n is 2.

A particularly interesting texaphyrin analog of the present invention isone having the structure: ##STR8## wherein M is a divalent metal ion andn is 1, or M is a trivalent metal ion and n is 2. In the above-describedmetallic complexes M may be a divalent metal ion selected from the groupconsisting of Ca⁺², Mn⁺², Co⁺², Ni⁺², Zn⁺², Cd⁺², Hg⁺², Sm⁺² and UO₂ ⁺²(and n is 1) In certain aspects M is preferably Cd⁺² or Zn⁺² or Hg⁺².When M is a trivalent metal ion, it is preferably selected from thegroup consisting of Mn⁺³, Co⁺³, Mn⁺³, Ni⁺³, Y⁺³, In⁺³, Pr⁺³, Nd⁺³, Sm⁺³,Eu⁺³, Gd⁺³, Tb⁺³, Dy⁺³, Er⁺³, Tm⁺³, Yb⁺³, Lu⁺³, and U⁺³ ; (and n is 2.)Most preferred trivalent metal ions are In⁺³, Y⁺³, Nd⁺³, Eu⁺³, Sn⁺³ andGd⁺³.

Additionally, the compounds of the present invention may be described ashaving the structure: ##STR9## wherein R and R' are F; R is H and R' isO(CH₂ CH₂ O)₂ CH₃ ; R is H and R' is SO₃ ⁻ ; or R is H and R' is CO₂ ⁻and M is a divalent metal ion and n is 1 or M is a trivalent metal ionand n is 2.

A particularly preferred texaphyrin-derived compound is one having thestructure: ##STR10## wherein M is a divalent metal ion and n is 1 or Mis a trivalent metal ion and n is 2.

In another aspect, the present invention involves a compound having thestructure: ##STR11## wherein M is a divalent metal ion and n is 1; or Mis a trivalent metal ion and n is 2 is exemplary of the texaphyrinderivatives of the present invention. So also is a compound having thestructure: ##STR12## wherein M is a divalent metal ion and n is 1 or Mis a trivalent metal ion and n is 2.

In yet another view, the present invention includes a compound havingthe structure: ##STR13## wherein R₁ and R₂ are H or CH₃ and M is Hg⁺²,Cd⁺², Co⁺² or Mn⁺², and n is 1; or M is Ln⁺³, Gd⁺³, Y⁺³ or In⁺³ and n is2; or R₁ is H, R₂ is Cl, Br, NO₂, CO₂ H or OCH₃, M is Zn⁺², Hg⁺², Sn⁺²or Cd⁺² and n is 1.

Absent metal ions, compounds of the present invention may have thestructure: ##STR14## or the structure: ##STR15## For example, a methodfor the synthesis of pentadentate expanded porphyrin compound is anaspect of the present invention. This method comprises synthesizing adiformyl tripyrrane; condensing said tripyrrane with an orthoaryldiamine 1,2-diaminoalkene or 1,2-diaminoalkane; and oxidizing thecondensation product to form a pentadentate expanded porphyrin compound.A preferred 1,2-diaminoalkene is diamisomaleonitrile. The orthoaryldiamine is preferably ortho phenylenediamine or a substituted orthophenylenediamine. Another preferred ortho aryldiamine is2,3-diaminonapthalene. Such a pentadentate expanded porphyrin compoundis complexed with a metal and wherein a metal complex is produced byreaction of the pentadentate expanded porphyrin compound with metalions.

The present invention also involves a method of deactivatingretroviruses and enveloped viruses in blood. This method comprisesadding a pentadentats expanded porphyrin analog metal complex asdescribed above to blood and exposing the mixture to light to facilitatethe formation of singlet oxygen.

Photodynamic tumor therapy comprising administering a pentadentateexpanded porphyrin analog complexed with a metal to a tumor host andirradiating the analog in proximity to the tumor is another aspect ofthe present invention.

A method for MRI enhancement comprising administering a diamagneticmetal ion (such as gadolinium, for example) complexed with texaphyrin ora texaphyrin derivative is also an aspect of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B show a schematic structural view of Texaphyrin (1)and various complexes (2, 3 and 4).

FIG. 2 shows a view of complex 4 (from FIG. 1B) showing pyridine andmacrocycle coordination to Cd. Ellipsoids scaled to the 40% probabilitylevel. The Cd ion lies in the plane of the nearly planar macrocycle(maximum deviation from planetary 0.10 (1) Å). Relevant Cd--N bondlengths (Å) are as follows: 2.418(7), N1; 2.268(8), NS; 2.505(7), N13;2.521(7), N20; 2.248(8), N23; 2.438(14), N1a; 2.473(12), N1b. SelectedN--Cd--N bond angles (deg) are as follows: N1-Cd-NS, 78.9(2); N1-Cd-N23,80.2(3); N8-Cd-N13, 68.4(2); N13-Cd-N20, 64.4(2); N20-Cd-N23, 68.2(3);N1a-Cd-N1b, 176.1(4).

FIG. 3 shows a view of complex 4 perpendicular to the plane through themacrocycle. Pyridine rings (not shown) lie perpendicular to themacrocycle with dihedral angles of 88.5 (4)o for ring a and 89.1 (3).for ring b.

FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D show a schematic representation ofthe reduced (1_(A)) and oxidized (2_(A)) forms of the free-base"texaphyrin" and representative five, six, and sere coordinate cadmiumcomplexes (3_(A) -5_(A)) derived from this "expanded porphyrin".

FIG. 5 shows a view of the cation 5_(aA) showing pyridine and macrocyclecoordination to Cd. Ellipsoids are scaled to the 30% probability level.The cadmium (II) cation lies in the plane of the nearly planarmacrocycle (max. deviation from planarity, 0.10(1) Å). Relevant Cd--Nbond lengths (Å) are: 2.418(7) N1; 2.268(8) N8; 2.505(7) N13; 2.521(7)N20; 2.248(8) N23; 2.438(14) N1a; 2.473(12) N1b. Selected N--Cd--N bondangles (o) are: 78.9(2) N1-Cd-N8; 80.2(3) N1-Cd-N23; 68.4(2) N8-Cd-N13;64.4(2) N13-Cd-N20; 68.2(3) N20-Cd-N23; 176.1(4) N1a-Cd-N1b. For furtherstructural details, see ref. 11.

FIG. 6 shows a view of cation 4_(bA) showing the atom labelling scheme.Thermal ellipsoids are drawn to 30% probability level. Relevant Cd--Nbond lengths (Å): N1 2.462(13); N2.254(9); N13 2.535(13); N20 2.526(12);N23 2.298(11); N1A 2.310(9). Selected N--Cd--N bond angles (o): N1-Cd-N878.3(4); N8-Cd-N13 67.8(4); N13-Cd-N20 64.1(4); N20-Cd-N23 67.3(4);N1A-Cd-macrocycle N angles range from 93.7(4) to 100.4(3)o. The nitratecounter anion (not shown) is not coordinated to the Cd atom.

FIG. 7 shows a view along the plane through the macrocycle illustratingthe face-to-face stacking of the cation 4_(bA) in the unit cell(macrocycle realted by 1-x,y,z). The macrocycle mean planes areseparated by 3.38 Å while the CdCd distance is 4.107(1) Å.

FIG. 8 shows a view of cation 4_(bA) perpendicular to the plane throughthe macrocycle (max. deviation 0.154(13) Å for C15). The Cd atom liesout this plane by 0.334(2) Å. BzIm (not shown) is oriented nearlyperpendicular to the macrocycle (dihedral angle of 86.3(3)o) and liesover the pyrrole ring defined by C22, N23, C24, C25, and C26.

FIG. 9A and FIG. 9B show a UV-visible spectrum of 3_(A) ·NO₃ 1.50×10⁻⁵ Min CHCl₃.

FIG. 10A and FIG. 10B show ¹ H NMR spectrum of 3_(A) ·NO₃ in CDCl₃. Thesignals at 1.5 and 7.26 ppm represent residual water and solvent peaksrespectively.

FIG. 11A and FIG. 11B show a low field region of the ¹ H NMR spectra (inCDCl₃) of 3_(A) ·NO₃ (spectrum A) and the bulk inhomogeneous materialfrom which crystals of complex 4b_(A) ·NO₃ were isolated (spectrum B).The signals marked `BzIm` ascribed to the bound benzimidazole ligand areobserved at 6.4, 6.81, and 7.27 ppm; the signals marked `s` are due toresidual solvent.

FIG. 12 shows ¹ H NMR spectral titration of 3_(A) ·NO₃ (initially6.85×10⁻³ M in CDCl₃) with increasing quantities of BzIm showingmoderate field region. The [BzIm]/[ligand] ratios are 0, 0.2, 0.6, 2.8,10, and 40 for traces A through F respectively, where [BzIm] and[ligand] represent the total molar concentration of added benzimidazoleand starting five-coordinate complex 3_(A) ·NO₃. The chemical shifts forthe BzIm signals in curve C (6.4, 6.62, 7.26 ppm) match well with thoseseen in the bulk sample from which the crystal of cation 4b_(A) wasisolated (c.f. FIG. 8B).

FIG. 13 shows the ¹ H NMR spectral titration of 3_(A) ·NO₃ (initially6.85×10⁻³ M in CDCl₃) with increasing quantities of BzIm showing changesoccurring in high field region. The [BsIm]/[ligand] ratios are 0, 0.2,0.6, 2.8, 10, and 40 for traces A through F respectively, where [BzIm]and [ligand] represent the total molar concentration of addedbenzimidazole and starting five-coordinate complex 3_(A) ·NO₃.

FIG. 14 shows changes in the ¹ H NMR chemical shift of the "meso" signalfor 3_(A) ·NO₃ plotted as a function of increasing [BzIm]. The terms[BzIm] and [ligand] represent the total molar concentration of addedbenzimidazole and starting five-coordinate complex 3_(A) ·NO₃.

FIG. 15 shows ¹ H NMR spectral titration of 3_(A) ·NO₃ (initially6.66×10⁻³ M in CDCl₃) with increasing quantities of pyridine showingchanges occurring in high field region. The [pyr]/[ligand] ratios are 0,5, 10, 14, 20, and 40 for traces A through F respectively. The terms[pyr] and [ligand] represent the total molar concentration of addedbenzimidazole and starting five-coordinate complex.

FIG. 16 shows changes in the ¹ H NMR chemical shift of the "meso" signalfor 3_(A) ·NO₃ as a function of increasing [pyr]. The terms [pyr] and[ligand] represent the total molar concentration of added benzimidazoleand starting five-coordinate complex 3_(A) ·NO₃.

FIG. 17A and FIG. 17B show metal complexes and derivatives (1_(B)-11_(B)) of compounds of the present invention.

FIG. 18A and FIG. 18B show the electronic spectrum of 2_(B) ·(OH)₂ inCHCl₃.

FIG. 19 schematically shows the structure, metal complexes andderivatives of compounds of the present invention (1_(C) -11_(C)).

FIG. 20A and FIG. 20B show the absorption spectrum of complex 1_(c) ·Clin deoxygenated methanol. FIG. 20B shows the fluorescence emissionspectrum recorded in this same solvent.

FIG. 21A and FIG. 21B show the triplet-triplet transient differencespectrum of 1_(C) ·Cl in deoxygenated methanol recorded 1 μs afterirradiation with a 10 ns pulse of 355 nm light (80 mJ). FIG. 21A showsthe rate of return to ground state, as monitored at 480 nm, andcorresponds to a triplet liftime of 67 μs.

FIG. 22 shows the spectral transmittance through human abdominal wallwith a thickness of 22-32 mm⁴⁷ (reference 47 taken from Example 5).

FIG. 23A, FIG. 23B, FIG. 23C and FIG. 23D show schematic structures ofpreviously developed prophyrin derivatives potentially useful asphotosensitizers. These include purpurins (1_(D)); verdins (2_(D));benz-fused porphyrins (3_(D)); and sulfonated phthalocyanines andnapthylocyanines (4_(D)).

FIG. 24A, FIG. 24B, FIG. 24C, FIG. 24D and FIG. 24E show schematicstructures of texaphyrin (5_(D)); sapphyrin (6_(D)); platyrin (7_(D));vinylogous porphyrin (8_(D)); and porphycenes (9_(D)).

FIG. 25A, FIG. 25B, and FIG. 25C show schematic structures of newaromatic tripyrroledimethine-derived macrocyclic ligands (10_(D)-16_(D)) analogous to texaphyrin (5_(D)).

FIG. 26 schematically (scheme 1) summarizes the synthesis of texaphyrin(5_(D)).

FIG. 27A, FIG. 27B, FIG. 27C and FIG. 27D show schematic structures ofproposed texaphyrin derivatives (23_(D) -28_(D)).

FIG. 28A and FIG. 28B show schematic structures of proposedmethane-linked texaphyrin derivatives (29_(D) and 30_(D)).

FIG. 29 shows mononuclear cell killing by complexes 1_(C) and 3_(A)without irradiation. Cell kill was determined by [3H]-Thy uptake afterphytohemagglutin (PHA) stimulation.

FIG. 30 shows mononuclear cell killing 1 μg/ml complex 3_(A) andirradiation. Cell kill was determined by [³ H]-Thy uptake after PHAstimulation.

FIG. 31A, FIG. 31B and FIG. 31C show expanded porphyrin-like macrocycleson hand. Alternatively to the pyrrolic hydrogen, a di- or trivalentcation such as Cd²⁺, Zn²⁺, In³⁺, etc. may be bound. In this case, thecomplex could bear a net overall charge, i.e. +1 for M⁺² and +2 for M⁺³.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention involves the synthesis of a novel "expandedporphyrin" system, 1_(B) (to which the trivial name "texaphyrin" hasbeen assigned), and includes description of the structure of thebispyridine adduct of its cadmium(II) complex. The presence in thisstructure of a near circular pentadentate binding core which is roughly20% larger than that of the porphyrins, coupled with the realizationthat almost identical ionic radii pertain for hexacoordinate Cd²⁺(r=0.92 Å) and Gd³⁺ (r=0.94 Å),²⁵ prompted exploration of the generallanthanide binding properties of this new monoanionic porphyrin-likeligand. The synthesis and characterization of a water-stablegadolinium(III) complex derived formally from a new 16,17-dimethylsubstituted analogue of the original "expanded porphyrin" system, aswell as the preparation and characterization of the correspondingeuropium(III) and samarium(III) complexes.

The aromatic "expanded porphyrin" system described herein provides animportant complement to the existing rich coordination chemistry ofporphyrins. For instance, by using methods similar to those described,zinc(II), manganese(II), mercury(II), and neodymium(III) complexes havebeen prepared and characterized.

The photophysical properties of this new series oftripyrroledimethine-derived "expanded porphyrins" ("texaphyrins") arereported; these compounds show strong low energy optical absorptions inthe 690-880 nm spectral range as well as a high triplet quantum yield,and act as efficient photosensitizers for the production of singletoxygen, for example, in methanol solution.

The present invention involves a major breakthrough in the area ofligand design and synthesis namely synthesis of the first rationallydesigned aromatic pentadentate macrocyclic ligand, atripyrroledimethine-derived "expanded porphyrin". This compound, towhich the trivial name "texaphyrin" has been assigned, is capable ofexisting in both its free-base form and of supporting the formation ofhydrolytically stable 1:1 complexes with a variety of metal cations,such as Cd²⁺, Hg²⁺, In³⁺, Y³⁺, Nd³⁺, Eu³⁺, Sm³⁺, and Gd³⁺, that are toolarge to be accommodated in a stable fashion within the 20% smallertetradentate binding core of the well-studied porphyrins. In addition,since the free-base form of texaphrin is a monoanionic ligand, thetexaphyrin complexes formed from divalent and trivalent metal cationsremain positively charged at neutral pH. As a result, many of thesecomplexes are quite water soluble--at least far more so than theanalogous porphyrin complexes.

The results to date, some of which are summarized herein, indicatestrongly that the expanded porphyrin-like macrocycles of the presentinvention should be efficient photosensitizers for the destruction offree HIV-1 and for the treatment of tumors in vivo and infectedmononuclear cells in blood. Altering the polarity and electrical chargesof side groups of these macrocycles is anticipated to alter markedly thedegree, rate, and perhaps site(s) of binding to free enveloped virusessuch as HIV-1 and to vitally-infected peripheral mononuclear cells.These substituent changes are also expected to modulate photosensitizertake-up and photosensitization of leukemia or lymphoma cellscontaminating bone-marrow as well as by normal cells of the marrow.

EXAMPLE 1

The porphyrins and related tetrapyrrole macrocycles are among the mostversatile of tetradentate ligands.¹ Attempts to stabilize highercoordination geometries, however, with larger porphyrin-like aromaticmacrocycles have met with little success.²⁻⁵ Indeed, to date, only theuranyl complex of "superphthalocyanine" has been isolated andcharacterized structurally,² although several other large porphyrin-likearomatic macrocycles, including the "sapphyrine",³,6 "oxosapphyrins",⁶,7"platyrins",⁸ "pentaphyrin",⁹ and "[26]porphyrin",¹⁰ have been preparedin their metal free forms. This example describes one aspect of thedevelopment of a new type of "expanded porphyrin" capable of binding avariety of metal cations. Also described herein is the originalsynthesis of compound²,11 an unprecedented porphyrin-like aromaticpentadentate ligand,²,12 and the structure of its cadmium (II)bispyridine complex 4. (See FIG. 1A and FIG. 1B for the structure ofcompound or complex 1-4)

The present involves preparation of the nonaromatic methylene-bridgedmacrocycle (compound 1) by the direct acid-catalyzed condensation of2,5-bis[3-ethyl-5-formyl-4-methylpyrrol-2-yl)methyl]-3,4-diethyl-pyrroleand orthophenylenediamine¹³ and determined it to be an ineffectivecheland.¹⁴ The present inventors have now found that stirring thereduced macrocyclic compound 1 with cadmium chloride for 24 hours inchloroform-methanol (1:2 v.v.) in the presence of air, followed bychromatographic purification on silica gel and recrystallization fromchloroform-hexanes, gives the cadmium(II) complex 3·Cl in 4% yield as adark green powder.¹⁵ Under the reaction conditions both ligand oxidationand metal complexation take place spontaneously.

The structure of compound 3 suggests that it can be formulated as eitheran 18 π-electron benzannelated [18]annulene or as an overall 22π-electron system; in either case an aromatic structure is defined. Theproton NMR spectrum of complex 3·Cl is consistent with the proposedaromaticity. For the most part, complex 3·Cl shows ligand features whichare qualitatively similar to those observed for compound 1. As would beexpected in the presence of a strong diamagnetic ring current, however,the alkyl, imine, and aromatic peaks are all shifted to lower field.Furthermore, the bridging methylene signals of compound 1 (at δ≅4.0)¹³are replaced by a sharp singlet, at 11.3 ppm, ascribable to the bridgingmethine protons. The chemical shift of this "meso" signal is greaterthan that observed for Cd(OEP)¹⁶ (δ≅10.0),¹⁷ an appropriate 18π-electron aromatic reference system, and is quite similar to thatobserved for the free-base form of decamethylsapphyrin (δ≅11.5- 11.7),³a 22 π-electron pyrrole-containing macrocycle.

The optical spectrum of complex 3·Cl bears some resemblance to those ofother aromatic pyrrole-containing macrocyles³,6,7,18 and providesfurther support for the proposed aromatic structure. The dominanttransition is a Soret-like band at 424 nm (ε=72,700), which isconsiderably less intense than that seen for Cd(OEP)(pyr)¹⁶ λ_(max) =421nm, ε=288,000).¹⁸ This peak is flanked by exceptionally strong N- andQ-like bands at higher and lower energies. As would be expected for alarger π system, both the lowest energy Q-like absorption (λ_(max)=767.5 nm, ε=41,200) and emission (λ_(max) =792 nm)) bands of complex3·Cl are substantially red-shifted (by ca. 200 nm!) as compared to thoseof typical cadmium porphyrins.¹⁸,19

When the above metal insertion was repeated with cadmium nitrate, acomplex was obtained in roughly 30% yield, which, on the basis ofmicroanalytical data,¹⁵ was formulated as the protonated complex 3·NO₃·(HNO₃). Upon treatment with excess pyridine and recrystallization fromchloroform-hexane, the bis-pyridine adduct complex 4-NO₃, with spectralproperties essentially identical with 3·Cl, was isolated as dark greencrystals.¹⁵ The molecular structure of 4-NO₃, determined by X-raydiffraction analysis, confirms the aromatic nature of the ligand (FIG.2).²⁰ The central five nitrogen donor atoms of complex 4 are essentiallycoplanar and define a near circular cavity with a center-to-nitrogenradius of ca. 2.39 Å (cf. FIG. 3), which is roughly 20% larger than thatfound in metalloporphyrins.²¹ The Cd atom lies in the plane of thecentral N₅ binding core. The structure of the "expanded porphyrin" 4thus differs dramatically from that of CdTPP¹⁶,22 or CdTPP-(dioxane)₂,²³in which the cadmium atom lies out of the porphyrin N₄ donor plane (by0.58 and 0.32 Å, respectively). Moreover, in contrast to cadmiumporphyrins, for which a five-coordinate square-pyramidal geometry ispreferred and to which only a single pyridine molecule will bind,²⁴ incomplex 4-NO₃ the cadmium atom is seven-coordinate, being complexed bytwo apical pyridine ligands. The configuration about the Cd atom is thuspentagonal bipyrimidal; a rare but not unknown geometry for cadmium(II)complexes.²⁵

Under neutral conditions complexes 3 and 4 appear to be more stable thancadmium porphyrins: Whereas treatment of CdTPP or CdTPP(pyr) withaqueous Na₂ S leads to cation loss and precipitation of CdS, in the caseof complexes 3 and 4 no demetallation takes place. (Exposure to aqueousacid, however, leads to hydrolysis of the macrocyle.) Indeed, it has notbeen possible to prepare the free-base ligand 2 by demetallation. Thetripyrroledimethine-derived free-base ligand 2 was synthesized directlyfrom 1 by stirring in air-saturated chloroform-methanol containingN,N,N'-tetramethyl-1,8-diaminonaphthalene.¹⁵ Although the yield is low(≦12%),²⁶ once formed, compound 2 appears to be quite stable: Itundergoes decomposition far more slowly than compound 1.¹³ Presumably,this is a reflection of the aromatic stabilization present in compound2. A further indication of the aromatic nature of the free-base"expanded porphyrin" 2 is the observation of an internal pyrrole NHsignal at δ=0.90, which is shifted upfield by over 10 ppm as compared tothe pyrrolic protons present in the reduced macrocyle 1.¹³ This shiftparallels that seen when the sp³ -linked macrocycle,octaethylporphyrinogen (δ(NH)=6.9),²⁷ is oxidized to the correspondingporphyrin, H₂ OEP (δ(NH)=3.74).¹⁷ This suggests that the diamagneticring current present in compound 2 is similar in strength to that of theporphyrins.

The aromatic "expanded porphyrin" system described herein provides animportant complement to the existing rich coordination chemistry ofporphyrins. For instance, by using methods similar to those described,zinc(II), manganese(II), mercury(II), and neodymium(III) complexes ofcompound 2¹⁵ have been prepared and characterized.

Literature citations in the following list are incorporated by referenceherein for the reasons cited.

REFERENCES

1. The Porphyrins; Dolphin, D., Ed.; Academic Press: New York,1978-1979; Vols. I-VII.

2. "Superphthalocyanine", a pentaaza aromatic phthalocyanine-like systemwas prepared by a uranyl-medicated condensation; it is not obtainable asthe free-base or in other metal-containing forms: (a) Day, V. W.; Marks,T. J.; Wachter, W. A. J. Am. Chem. Soc. 1975, 97, 4519-4527. (b) Marks,T. J.; Stojakovic, D. R. J. Am. Chem. Soc. 1978, 100, 1695-1705. (c)Cuellar, E. A.; Marks, T. J. Inorg. Chem. 1981, 208, 3766-3770.

3. Bauer, V. J.; Clive, D. R.; Dolphin, D.; Paine, J. B. III; Harris, F.L.; King, M. M.; Loder, J.; Wang, S.-W. C.; Woodward, R. B. J. Am. Chem.Soc. 1983, 105, 6429-6436. To date only tetracoordinated metal complexeshave been prepared from these potentially pentadentate ligands.

4. For an example of a porphyrin-like system with a smaller centralcavity, see: (a) Vogel, E; Kocher, M.; Schmickler, H.; Lex, J. Angew.Chem. 1986, 98, 262-263; Angew. Chem., Int. Ed. Engl. 1986, 25, 257-258.(b) Vogel, E.; Balci, M.; Pramod, K.; Koch, P.; Lex, J.; Ermer, O.Angew. Chem. 1987, 99,909-912; Angew. Chem., Int. Ed. Engl. 1987, 26,928-931.

5. Mertes et al. have recently characterized a five-coordinate coppercomplex of an elegant (but nonaromatic) porphyrin-like "accordion"ligand derived from dipyrromethines: (a) Acholla, F. V.; Mertes, K. B.Tetrahedron Lett. 1984, 3269-3270. (b) Acholla, F. V.; Takusagawa, F.;Mertes, K. B. J. Am. Chem. Soc. 1985, 6902-6908. Four-coordinate coppercomplexes of other nonaromatic pyrrole-containing macrocycles have alsobeen prepared recently: Adams, H.; Bailey, N. A.; Fenton, D. A.; Moss,S.; Rodriguez de Barbarin, C. O; Jones, G. J. Chem. Soc., Dalton Trans.1986, 693-699.

6. Broadhurst, M. J.; Grigg., R; Johnson, A. W. J. Chem. Soc., PerkinTrans. 1 1972, 2111-2116.

7. (a) Broadhurst, M. J.; Grigg, R.; Johnson, A. W. J. Chem. Soc., Chem.Commun. 1969, 23-24. Broadhurst, M. J.; Grigg, R.; Johnson, A. W. J.Chem. Soc., Chem. Commun. 1969, 1480-1482. Broadhurst, M. J.; Grigg, R.;Johnson, A. W. J. Chem. Soc., Chem. Commun. 1970, 807-809.

8. (a) Berger, R. A.; LeGoff, E. Tetrahedron Lett. 1978, 4225-4228. (b)LeGoff. E.; Weaver, O. G. J. Org. Chem. 1987, 710-711.

9. (a) Rexhausen, H.; Gossauer, A. J. Chem. Soc. Chem. Commun. 1983,275. (b) Gossauer, A. Bull. Soc. Chim. Belg. 1983, 92, 793-795.

10. Gosmann, M.; Franck, B. Angew. Chem. 1986, 98, 1107-1108; Angew.Chem., Int. Ed. Engl. 1986, 25, 1100-1101.

11. The systematic name for compounds 2 is4,5,9,24-tetraethyl-10,23-dimethyl-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19]heptacosa-1,3,5,7,9,11(27),12,14,16,18,20,22(25),23-tridecaene.

12. Nonaromatic planar pentadentate pyridine-derived ligands are known.See, for instance: (a) Curtis, N. F. In Coordination Chemistry ofMacrocyclic Compounds; Melson, G. A., Ed.; Plenum: New York, 1979;Chapter 4. (b) Nelson, S. M. Pure Appl. (Chem. 1980, 52, 2461-2476. (c)Ansell, C. W. G.; Lewis, J.; Raithby, P. R.; Ramsden, J. N.; Schroder,M. J. Chem. Soc., Chem. Commun. 1982, 546-547. (d) Lewis, J.;O'Donoghue, T. D.; Raithby, P. R. J. Chem. Soc., Dalton Trans. 1980,1383-1389. (e) Constable, E. C.; Chung, L.-Y.; Lewis, J.; Raithby, P. R.J. Chem. Soc., Chem. Commun. 1986, 1719-1720. (f) Constable, E. C.;Holmes, J. M.; McQueen, R. C. S. J. Chem. Soc., Dalton Trans. 1987, 5-8.

13. Sessler, J. L.; Johnson, M. R..; Lynch, V. J. Org. Chem. 1987, 52,4394-4397.

14. Sessler, J. L.; Johnson, M. R.; Lynch, V.; Murai, T. J. Coord.Chem., in press.

15. Satisfactory spectroscopic, mass spectrometric, and/or analyticaldata were obtained for all new compounds.

16. OEP=octaethylporphyrin and TPP=tetraphenylporphyrin; the prefixes H₂and Cd refer to the free-base and cadmium(II) forms, respectively;pyr=pyridine.

17. (a) Scheer, H.; Katz, J. J. In Porphyrins and Metalloporphyrins;Smith, K., Ed.; Elsevier: Amsterdam, 1975; Chapter 10. (b) Janson, T.R.; Katz, J. J.; in ref. 1, Vol. IV, Chapter 1.

18. Gouterman, M., in ref. 1, Vol. III, Chapter 1.

19. Becker, R. S.; Allison, J. B. J. Phys. Chem. 1963, 67, 2669.

20. Crystal data: 4·NO₃ crystallized from CHCl₃ -hexanes in thetriclinic space group, P1 (no. 1), with a=9.650 (3) Å, b=10.217 (4) Å,c=11.295 (4) Å, a=98.16 (3), β=107.05 (2), γ=92.62 (3)°, V=1049.3 (6)Å³, and ρ_(c) =1.49 g-cm⁻³ for Z=1. Unique reflections (5654)(4936 withF≧6σ(F)) using ω scans were collected at 193K on a Nicolet R3m/V with MoKα radiation (λ=0.71069 Å) out to 2θ of 50°. Data corrected for decay,Lp effects, and absorption. Refined by conventional means to anR=0.0534. All non-H atoms refined anisotropically. H atom positionscalcualted (d_(C-H) 0.96 Å) and refined isotropically riding on therelevant C atom. The non-coordinated nitrate ion is within H-bondingdistance of the CHCl₃ solvent molecule with O . . . C (CHCl₃) and O . .. H distances of 3.00(2) Å and 2.46(2) Å, respectively: For full detailssee Supplementary Material.

21. Hoard, J. L., in ref. 17a, Chapter 8.

22. Hazell, A. Acta Crystallogr., Sect. C: Cryst. Struct. Commun. 1986,C42, 296-299.

23. Rodesiler, P. F.; Griffith, E. H.; Ellis, P. D.; Amma, E. L. J.Chem. Soc. Chem. Commun. 1980, 492-493.

24. (a) Miller, J. R. Dorough, G. D. J. Am. Chem. Soc. 1952, 74,3977-3981. (b) Kirksey, C. H.; Hambright, P. Inorg. Chem. 1970, 9,958-960.

25. Compound 4 appears to be the first seven-coordinate cadmium complexderived from all nitrogen donors. For examples of other pentagonalbipyramidal cadmium complexes, see: (a) Cameron, A. F.; Taylor, D. W.;Nuttall, R. H. J. Chem. Soc., Dalton Trans. 1972, 1608-1614. (b) Liles,D. C.; McPartlin, M.; Tasker, P. A.; Lip, H. C.; Lindoy, L. F. J. Chem.Soc., Chem. Commun. 1976, 549-551. (c) Nelson, S. M.; McFall, S. G.;Drew, M. G. B.; Othman, A. H. B.; Mason, N. G. J. Chem. Soc. Chem.Commun. 1977, 167-168. (d) Drew, M. G. B. Othman, A. H. B.; McFall, S.G.; McIlroy, A. D. A.; Nelson, S. M. J. Chem. Soc., Dalton Trans. 1977,1173-1180. (e) Charles, N. G. Griffith, E. A. H.; Rodesiler, P. F.;Amma, E. L. Inorg. Chem. 1983, 22, 2717-2723.

26. Other oxidants, including DDQ, Ag₂ O, I₂, PtO₂, PbO₂ SeO₂, and Ph₃CBF₄, either failed to react or gave rise only to decompositionproducts.

27. Whitlock, H. W., Jr.; Buchanan, D. H. Tetrahedron Lett. 1969, 42,3711-3714.

EXAMPLE 2

Although the porphyrins and related tetrapyrrolic compounds remain amongthe most widely studied of all known macrocycles, ¹ relatively littleeffort has been devoted to the development of larger conjugated pyrrolecontaining systems.²⁻¹² Large, or "expanded" porphyrin-like systems,however, are of interest for several reasons: They could serve aspossible aromatic analogues of the better studied porphyrins²⁻⁸ or serveas potential biomimetic models for these or other naturally occurringpyrrole-containing systems.¹³,14 In addition, large pyrrole containingsystems offer exciting possibilities as novel metal bindingmacrocyles.²,9-12,15 For instance, suitably designed systems could actas versatile ligands capable of binding larger metal cations and/orstabilizing higher coordination geometries²,16 than those routinelyaccommodated within the normally tetradentate ca. 2.0 Å radius porphyrincore.¹⁷ The resulting complexes could have important application in thearea of heavy metal chelation therapy or as new vehicles for extendingthe range and scope of coordination chemistry.¹⁵,18 In recent years anumber of potentially pentadentate polypyrrolic aromatic systems,including the "sapphyrins",³,4 "oxosapphyrins",⁵ "smaragdyrins",³,4platyrins,⁶ and "pentaphyrin"⁷ have been prepared and studied as theirmetal-free forms. For the most part, however, little or no informationis available for the corresponding metallated forms. Indeed, the uranylcomplex of "superphthalocyanine" was the only metal-containingpentapyrrolic system which has been prepared and characterizedstructurally.² Unfortunately, the "superphthalocyan" system isapparently not capable of existence in either its free-base or othermetal-containing forms.² Thus, prior to the present invention, noversatile, structurally characterized, pentadentate aromatic ligandsware available,¹¹ although a number of nonaromatic pyridine-derivedpentadentate systems had previously been reported.¹⁹,20 The aspect ofthis invention described here further shows development of a new classof pyrrole-derived aromatic "expanded porphyrins" capable of binding avariety of metal cations and stabilizing a range of unusual coordinationgeometries. The present inventors have recently communicated thesynthesis of compound 2_(A),¹¹ (see Example 1) an unprecedentedporphyrin-like monoanionic aromatic pentadentate ligand to which hasbeen assigned the trivial name "texaphyrin" (for large Texas-styleporphyrin),¹⁸ and the structure of its seven coordinate cadmium(II)bispyridine pentagonal bipyramidal complex 5a_(A). Due to the importanceof cadmium complexes in possible chelation based therapeuticapplications²¹,22 and as potential structural probes for naturalmetalloproteins (e.g. employing ¹¹³ Cd NMR spectroscopy),²³ thecoordination properties of the cadmium-containing "texaphyrin" systemwere investigated further. The present example reports thecharacterization by single crystal X-ray diffraction analysis of amonoligated six coordinate cadmium(II) benzimidazole pentagonalpyramidal cationic complex 4b_(A) corresponding formally to acoordinatively unsaturated analogue of 5a_(A). The present description,which includes the results of solution base binding (K_(eq).) studiesfor both pyridine (pyr) and benzimidazole (BzIm), constitutes the firststructurally documented instance wherein the same macrocyclic ligand hasbeen used to support these two rare, but not unknown,¹⁹ coordinationgeometries about the same metal cation.²⁴ See FIG. 4A, FIG. 4B, FIG. 4Cand FIG. 4D for the schematic structure of compounds and complexes ofthe present invention referred to here as 1_(A), 2_(A), 3_(A), 4a_(A),5a_(A) and 5b_(A).

Treatment of the reduced, sp³ form of the macrocyle (1_(A))¹⁴ withcadmium chloride or cadmium nitrate in air-saturated chloroform-methanolleads in both cases to the formation of green solutions. Followingchromatographic purification on silica gel and recrystallisation fromchloroform-hexanes, the five-coordinate "texaphyrin" chloride or nitratecomplexes 3_(A) ·Cl and 3_(A) ·NO₃ were obtained in analytically pureform (as the demihydrates) in roughly 25% yield. When, however, themetal insertion procedure was carried out (using cadmium nitrate) underreaction and purification conditions identical to those described above,with the exception that chromatography was effected on SEPHADEX, amixtures of crystalline and noncrystalline green solids was obtained.Treatment of this apparently inhomogeneous bulk material, which failedto analyze as a pure five coordinate complex, with excess pyridine andrecrystallization from chloroform-hexane produced the bispyridinecomplex 5a_(A) -NO₃ as dark green crystals in essentially quantitativeyield. As communicated earlier,¹¹ (see Example 1) an X-ray crystaldiffraction analysis served to confirm the pentagonal bipyramidalcoordination geometry postulated for this bisligated seven coordinatecomplex and the planar pentadentate nature of the macrocyclic"texaphyrin" ligand 2_(A) (c.f. FIG. 5).

As a first step towards determining the nature of the above intermediateproduct, a single crystal was isolated from the inhomogeneaus solidmixture and subject to an X-ray diffraction analysis. The structure soobtained (FIG. 6) was quite unexpected: It revealed a six coordinatepentagonal pyramidal cadmium(II) complex (4b_(A) ·NO₃) wherein one ofthe two possible axial ligation sites is occupied by a boundbenzimidazole (BzIm) with the nitrate counter anion not beingcoordinated to the central Cd atom. The first donor nitrogens of thepentadentate "texaphyrin" macrocycle then serve to complete thecoordination sphere about cadmium. As shown in FIG. 6, the 5 donor atomsof the ligand are bound to the Cd atom which lies out of the plane ofthe macrocycle being displaced by 0.338(4) Å from the N₅ donor planetowards the coordinated nitrogen of the benzimidazole ligand. This outof plane distance, which is similar to that seen in CdTPP-(dioxane)₂ ²⁵(0.32 Å)²⁶ (but smaller than that observed in CdTPP²⁷), is in markedcontrast to that observed for the corresponding bispyridine pentagonalbipyramidal adduct 5a_(A) -NO₃.¹¹ In this earlier structure, thecadmium(II) cation was found to lie essentially within the plane ofmacrocycle (c.f. FIG. 5). Cation 4b_(A) further differs from 5a_(A) inthat, within the crystal lattice, two molecules stack on one another ina face-to-face fashion (FIG. 7) being separated by van der Waals'distances of ca. 3.38 Å. As a result, the alkyl groups in any givenmolecule are all displaced to the BzIm-bearing side of the macrocyclicplane. In common with the bispyridine structure,¹¹ however, in cation4b_(A) the sp² atoms of the macrocycle are all essentially planar (FIG.8) with the maximum deviation from planarity (0.154(13) Å) being foundfor C11. Also in common with complex 5a_(A) -NO₃, the five ligandnitrogens define a near circular binding cavity with acenter-to-nitrogen radius of ca. 2.42 Å, which is roughly 20% largerthan that found in metalloporphyrins.¹⁷

The above structural results support the original formulation of"texaphyrin" 2_(A) as large 22 π-electron (or benzannelated 18π-electron) aromatic porphyrin-like ligand.²⁸ They also clearlydemonstrate that this "expanded porphyrin" is capable of supporting morethan one kind of "unusual" coordination geometry about cadmium.

The above structural results also provide insight into the nature of theinhomogeneous cadmium-containing intermediate obtained following metalinsertion and sephadex-based purification: At least a portion of thismaterial consists of the six-coordinate BzIm ligated complex 4b_(A)·NO₃. Although it is certainly plausible to postulate that thecoordinated BzIm in cation 4b_(A) derives from ligand degradationreactions associated with metal insertion and accompanying oxidation(presumably involving electrophilic aromatic deacylation of antripyrrane α-carbon and subsequent condensation with ortho-phenylenediamine), the observation of this six-coordinate species does notestablish unambiguously that such BzIm coordination is chemicallyreasonable. This point is of particular interest since in the presenceof excess pyridine, it is the bisligated seven coordinate cationicspecies 5a_(A) which is favored in the solid state. It was believed tobe of importance to determine the solution binding properties ofcompound 3_(A) ·NO₃ in the presence of both benzimidazole and pyridine.The objective was not only to probe the ligation differences (if any) ofthese two axial bases but also to define further the nature of theintermediate inhomogeneous solid material formed following Cd insertionand SEPHADEX-based purification, testing in particular the reasonableassumption that this material consists of a mixture of the five and sixcoordinate cations 3_(A) and 4b_(A).

For a rigorously five coordinate starting cadmium complex, such as thatrepresented schematically by structure 3_(A), where neither the counteranion nor adventitious ligands serves to occupy an apical coordinationsite, base binding can be considered to occur in accord with equations(1) and (2) shown below. Under conditions where K₁ ≧K₂, these processescan be considered to occur sequentially, giving first a monoligated,presumably pentagonal pyramidal six coordinate species (such as 4b_(A)),followed by a coordinatively saturated bisligated pentagonal bipyramidalproduct akin to 5a_(A). Where, however, K₂ >>K₁ this stepwise conceptualapproach is invalid. Under these conditions, it becomes easier toanalyze the base binding in terms of direct formation of the bisligatedmaterial as shown in equation (3).

    L+B LB K.sub.1 =[LB]/[L][B]                                (1)

    LB+B LB.sub.2 K.sub.2 =[LB.sub.2 ]/[LB][B]                 (2)

    L+2B LB.sub.2 K.sub.1 K.sub.2 =[LB.sub.2 ]/[L][B].sup.2    (3)

In the context of the present study therefore the problem becomes one offinding a solution-based analytical method that will allow changesassociated with mono and bis ligation to be probed, and using theaccompanying changes to determine as appropriate K₁, K₂, or K₁ K₂.

Optical spectroscopy is an important method of characterization fornonlabile metal complexes. In cases where absorption changes accompanyligand binding, optical spectroscopy also provides a convenient means ofdetermining base binding constants.²⁹ In the case of cadmium tetraphenylporphyrin (CdTPP), for instance, Miller and Dorough,³⁰ by monitoring thechanges associated with the two low energy Q bands of the absorptionspectrum, determined a value for the binding of a single pyridine axialligand to the unligated four-coordinate starting metalloporphyrin inbenzene at 29.9° (K₁) of roughly 2,700M⁻¹. Interestingly, these³⁰ andlater workers³¹ obtained no evidence for the formation of a bisligatedCdTPP-(pyr)₂ ²⁵ species. Thus, although a pseudo octahedral coordinationgeometry is defined in the solid state by the weakly bound axial ligandsof CdTPP-(dioxane)₂,²⁶ there is no evidence that such a structure isattained in pyridine-containing benzene solution.

The optical spectrum of purified complex 3_(A) ·NO₃ (FIG. 9A and FIG.9B) bears some elements in common with that of cadmium porphyrins.³⁰⁻³⁴For instance, complex 3_(A) ·NO₃ in CHCl₃ displays a strong Soret-likehigh energy transition at 425 nm (ε=82,800) which is considerably lessintense than that seen in cadmium porphyrins (e.g. CdOEP:²⁵ λ_(max)(CHCl₃ /MeOH v/v 19/1)=406 nm, ε=272,000).³⁵ This complex also displaysexceptionally strong flanking N- and Q-like bands at higher and lowerenergy. The lowest energy Q-like band (λ_(max) =770 nm, ε=49,800) isparticularly noteworthy: It is shifted to the red by ca. 200 nm and isalmost a factor of four more intense than the lowest energy Q-typetransition seen in typical cadmium porphyrins (e.g. CdOEP: λ_(max)(CHCl₃ /MeOH v/v 19/1)=571 nm, ε=15,400).³⁵ We consider such behavior tobe reflective of the larger delocalized aromatic system present in theoverall 22 π-electron "texaphyrins" than in the 18 π-electronporphyrins. Interestingly, the lowest energy transition seen in thecadmium complex of3,8,12,13,17,22-hexaethyl-2,7,18,23-decamethylsapphyrin in CHCl₃ is 701nm,³⁵ whereas that seer for the uranyl complex of "superphthalocyanine"is 914 nm.²⁶ Thus the lowest energy transition of 3_(A) ·NO₃ liesintermediate in energy between those observed for these for verydifferent 22 π-electron pentapyrrolic reference systems.

Unfortunately, in spite of the gross qualitative resemblance between theoptical spectrum of 3_(A) ·NO₃ and the other pyrrole-containing aromaticmacrocycles described above, optical spectroscopy has proved to be anineffective means of determining axial ligation properties of cation3_(A). For instance, addition of excess pyridine to a solution of 3_(A)·NO₃ in CHCl₃ caused only a ca. 1.5 nm red shift in the Soret-like bandand a 3.5 nm blue shift of the lowest energy Q-type band. (Similarinsignificant changes are also observed upon BzIm addition.) Thus, atleast in the case of the cadmium complexes, the optical properties ofthe "texaphyrin" expanded porphyrin system appear to be largelydetermined by the overall macrocyclic skeleton and relativelyinsensitive to changes in the electron environment of the bound cation.

Cadmium(II) complexes of "texaphyrin" 2_(A) are diamagnetic and hencereadily susceptible to study by ¹ H NMR methods. As shown in FIG. 10Aand FIG. 10B, the ¹ H NMR of 3_(A) ·NO₃ shows general features which aretypical of those expected for a large aromatic pyrrole-containingmacrocycle.³⁶ For instance, as compared to the sp³ form of the ligand(1)¹⁴ the alkyl, imine, and aromatic peaks are all shifted to lowerfield. Even more diagnostic, however, are the presence of "meso" signalsascribable to the bridging sp² hybridized methine protons in both thefree base "texaphyrin" 2 and its various cadmium containing derivatives3-5. These bridging protons resonate at ca. 7 ppm lower field than thecorresponding bridging methylene signals of the original sp³ form of theligand (1_(A)).¹⁴ In fact, the "meso" signals of 3_(A) ·NO₃ is foundroughly 1 ppm down field from those of typical β-alkyl substitutedcadmium porphyrins (e.g. Cd(OEP),²⁵,36 δ≅10.0) and approach in value thechemical shifts observed for diamagnetic sapphyrins (e.g., for free-basedecamethylsapphyrine,³ δ≅11.5-11.7). Such observations are notunexpected in light of the highly delocalized π character postulated forthe 22 π-electron "texaphyrin" systems.

FIG. 11A and FIG. 11B provide a comparison of the low field region ofthe ¹ H NMR spectra of 3_(A) ·NO₃ and the crude material from which thecrystals of cation 4b_(A) were obtained. The most striking differencebetween these two spectra is the presence of a small broad signal at ca.6.4 ppm and two sharper, more pronounced peaks at 6.81 and 7.27 ppm inthe spectrum of the bulk material (FIG. 11B). Although it is tempting toassign these features as signals arising from bound BzIm present incation 4b_(A), this conclusion is not necessarily obvious: Thecarbon-bound protons of free BzIm in CDCl₃ resonate at 7.25 (m, 2H),7.75 (m, 2H), and 8.41 (s, 1H) ppm.³⁷ Although shifts to higher field isexpected on binding to cation 3_(A), it is not clear that the expectedchanges would be as large as those actually observed. A completespectral titration of complex 3_(A) ·NO₃ with BzIm was thereforeundertaken in an effort to address this matter and to assignunambiguously the 6.4, 6.81, and 7.27 ppm signals. The results of thesetitrations are given in FIG. 12 and FIG. 13.

A striking feature of the ¹ H MR titration shown in FIG. 12 is thedramatic change in chemical shift that occurs for the BzIm signals uponcomplexation to cation 3_(A). Equally important, however is theobservation that the qualitative features of the bulk cadmium containingmaterial discussed above (c.f. FIG. 11B) are reproduced upon theaddition of roughly 3/5 equivalents of BzIm to purified 3_(A) ·NO₃ !This dramatic result provides, in our estimation, unambiguous supportfor the structural assignment of cation 4b_(A) made on the basis ofX-ray diffraction analysis. It also confirms qualitatively the originalsupposition that the inhomogeneous material isolated after Cd insertionand Sephadex purification does indeed involve an admixture of five andsix coordinated species (i.e. 3_(A) ·NO₃ and 4b_(A) ·NO₃).

For quantitative K_(eq). determinations it proved easiest to monitor thechanges associated with the "meso" signals. Here, sharp peaks,indicative of fast ligand exchange,²⁹,38 and reasonably large changes inchemical shift were observed (FIG. 13). In addition, no interferingBzIm-based resonances are found in this region. In FIG. 14 the changesin chemical shift for the "meso" protons in complex 3_(A) ·NO₃ areplotted as a function of added BzIm. The resulting titration curveshows, that at least for this base, axial ligation can be considered asoccurring in two essentially independent stepwise binding processes.Standard analysis³⁸ of the data at both very low and very highconversion gave values of K₁ =1.8±0.2×10⁴ and K₂ =13±3.

Just as was the case for BzIm, addition of pyridine to the fivecoordinate complex 3_(A) 19 NO₃ gave rise to easily detected andwell-defined changes in the chemical shift of the "meso" signals (FIG.15). In marked contrast to the results obtained with BzIm, however,ligation in this case cannot be considered to be occurring in a discretestepwise manner. This is quite apparent from an inspection of FIG. 13 inwhich the changes in chemical shift for the "meso" protons in complex3_(A) ·NO₃ are plotted as a function of increasing pyridineconcentration. Analysis of this binding isotherm using standardmethods³⁸ then gave values of K₁ ≅1.6M⁻¹ and K₁ K₂ =315±30M⁻².

The above K₁ and K₂ (or K₁ K₂) values are predicated on the assumptionthat the cadmium complexes under discussion are stable with regards todemetallation and that the equilibria of equations 1 and 2 (or 3)pertain under the conditions of base binding. The first of theseconcerns is readily apparent: If demetallation occurs, then obviouslyone is not studying base binding! All control experiments, however,suggested that the cadmium complexes derived from the "texaphyrin"ligand are many orders of magnitude more stable than those of theconsiderably smaller porphyrins. In fact, demetallation does not occureven when the complexes are challenged with excess sulfide anion (whichserves to demetallate CdTPP²⁵,35);³⁹ it thus appears unlikely that sucha process will occur in the presence of pyridine or benzimidazole. Thesecond concern is particularly important within the context ofquantitative work: If, for instance, the starting complex 3·NO₃ is notrigorously five coordinate, then K₁ (and perhaps K₂ as well) wouldrepresent an axial ligand displacement reaction rather than a pureaddition process as implied above. Control experiments indicate that theassumption of initial five coordination is reasonable: Independenttitrations of 3_(A) ·NO₃ with NH₄ NO₃ and H₂ O indicate that only modestand monotonic changes in the chemical shift of the "meso" signals takeplace over the course of adding ≧50 equivalents of these potentiallyadventitious ligands.⁴⁰ This means that either "complete" binding occursat 1:1 stoichiometry (essentially ruled out in the case of H₂ O on thebasis of analytical data), or that these species are poorly coordinatingin CHCl₃ so that five coordination pertains about cadmium; the latterinterpretation appearing more likely.

To the extent that the above assumptions are valid, the K_(eq). valuesobtained for BzIm and pyr binding in solution provide an accuratereflection of the coordination behavior observed in the solid state. Forinstance, at the concentrations used for the ¹ H NMR titrationexperiments (ca. 5×10⁻³ M) complex 3_(A) ·NO₃ will be roughly 20%converted to the six coordinate form (4b_(A) ·NO₃) following theaddition of only 0.2 molar equivalents of BzIm, and 90% convertedafter-the addition of 1.0 molar equivalent. Interestingly, even in thepresence of 10 molar equivalents, the resulting monoligated species4b_(A) will only be 35% converted to the corresponding bisligated sevencoordinate form (5b_(A)). Thus for benzimidazole a large concentrationrange pertains in solution wherein in the monoligated cationic complex4b_(A) is the dominant species. The equilibrium data also showed,however, that in solution it will always be either the bisligatedspecies 5a_(A) or unligated starting complex 3_(A) which dominates inthe presence of excess pyridine. For instance, under the conditions ofthe ¹ H NMR titrations, complex 3_(A) ·NO₃ will be roughly 5% convertedto pentagonal bipyramidal product 5a_(A) -NO₃ after the addition of 3equivalents of pyridine and roughly 35% converted to this species afterthe addition of 10 equivalents.

Both static and electronic factors may be invoked to explain thedifferent ligation properties for pyridine and benzimidazole.Considerable work with metelloporphyrins, particularly in the context ofheme model chemistry,⁴¹ has served to establish the strongercoordinating abilities of imidazole type ligands relative topyridine-type bases, an observation that is generally ascribed to thepoorer π basicity of the latter systems.^(41a),42 Thus the high K₁ value(relative to pyridine) observed for BzIm binding to cation 3_(A) comesas little surprise. What is more puzzling, however, is the observationthat K₂ for this base is so low: At first glance it appears unreasonablethat monoligation would be stable in the presence of this stronger πbase since preferential conversion to the coordinatively saturated sevencoordinate species occurs in the presence of pyridine. An inspection ofthe crystal structure shown in FIG. 6, however, provides the basis foran explanation: The BzIm residue lies nearly perpendicular to themacrocycle in 4b_(A) and is oriented over the pyrrole ring containingN23. As a result, H8A of the BzIm base is in close proximity to severalatoms of this ring, making close contacts (Å) with N23 (2.65(2)), C24(2.69(2)), and C22 (2.81(2)). Thus, as has been well-documented in thecase of heme models and encumbered imidazoles,^(41b),43 steric hindranceappears to be the fundamental factor favoring 6-coordination in thepresence of excess BzIm. Thus both steric and electronic effects serveto differentiate the ostensibly very different binding behavior of BzImand pyr in the present "expanded porphyrin" system. Such effects alsoprovide inter alia a rationale for the formation and selectiveisolation, in the solid state, of complexes 4b_(A) ·NO₃ and 5a_(A) ·NO₃.

The pentadentate 22 π-electron porphyrin-like "texaphyrin" macrocycle isan effective and versatile ligand for cadmium(II). It is capable ofsupporting the formation of three rare coordination geometries for thiscation, namely, pentagonal, pentagonal pyramidal, and pentagonalbipyramidal. Whereas the first of these forms is currently only inferredon the basis of analytical and solution phase studies, the latter twogeometries have been characterized both in solution and in the solidstate by single crystal X-ray diffraction analyses. The "texaphyrin"system thus represents, to the best of our knowledge, the firststructurally documented system capable of supporting both pentagonalpyramidal and pentagonal bipyramidal geometries about the same centralmetal cation. This unique cheland also endows these cadmium complexeswith several other important properties. These include an opticalspectrum with an unusually low energy Q-type band, and a stability withregards to demetallation which far exceeds that of the correspondingcadmium(II) porphyrins. The first of these properties suggests that thepresent "texaphyrin" (2_(A)) or other "expanded porphyrin" systemsshould find important application in the areas of photodynamic therapyor photosynthetic modelling studies where low energy absorptionproperties would be beneficial.⁴⁴ The second property suggests thatsystems similar to those presently described might provide the basis forthe development of effective chelation-based detoxification therapiesfor cadmium, a metal presently ranking only behind mercury and lead intoxicological importance,²¹ and one for which few, if any, therapiescurrently exist.²²

Electronic spectra were recorded on a Beckman DU-7 spectrophotometer.Proton and ¹³ C NMR spectra were obtained in CDCl₃ using CHCl₃ (δ=7.26ppm for ¹ H ; 77.0 ppm for ¹³ C) as an internal standard. Proton NMRspectra were recorded on either Nicolet NT-360 (360 MHz), or GeneralElectric QE-300 (300 MHz) spectrometer. Carbon spectra were measured at125 MHz using the Nicolet NT-500 spectrometer. Fast atom bombardmentmass spectrometry (FAB MS) was performed using a Finnigan-MAT TSQ-70instrument and 3-nitrobenzyl alcohol as the matrix. Elementary analyseswere performed by Galbraith Laboratories. X-ray structures were solvedas described below and in references 11 and 14.

All solvents and reagents were of reagent grade quality, purchasedcommercially, and used without further purification. Sigma lipophilicSephadex (LH-20-100) and Merck type 60 (230-400 mesh) silica gel wereused for column chromatography. The, sp³ form of the ligand (1_(A)) wasprepared in ≧90% yield using the acid catalyzed method describedearlier.¹⁴ The currently higher yield does not derive from a fundamentalchange in procedure but simply reflects a greater experience with thisparticular key reaction.

Preparation of4,5,9,24-tetraethyl-10,23-dimethyl-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19] heptacosa-1,3,5,7,9,11(27),12,14,16,18,20,22(25),23-tridecaene,free-base "texaphyrin" 2_(A). Macrocycle 1_(A) ¹⁴ (50 mg, 0.1 mmol) wasstirred in methanol/chloroform (150 ml, v/v 2/1) in the presence ofN,N,N',N'-tetramethyl-1,8-diaminonaphthalene ("proton sponge") for oneday at room temperature. The reaction mixture was then poured into icewater. The organic layer was separated and washed with aqueous ammoniumchloride solution and then brine. Following concentrations on a rotaryevaporator, the crude material was purified by chromatography onSEPHADEX using first pure chloroform and then chloroform/methanol(v/v/10/1) as eluents. After several faster red bands were discarded, adark green band was collected, concentrated in vacuo, and recrystallizedfrom chloroform/n-hexane to give the sp² form of the ligand as a darkgreen powder in yields ranging from 3-12% with the better yields onlybeing obtained on rare occasions. For 2_(A) : ¹ H-NMR (CDCl₃): δ=0.90(1H, br.s, NH), 1.6-1.8 (12H, m, CH₂ CH₃), 3.05 (6H, s, CH₃), 3.42-3.58(8H, m, CH₂ CH₃), 8.25 (2H, m, phen. CH), 9.21 (2H, s, CH═N, 9.45 (2H,m, phen. CH), 11.25 (2H, s, CH═C); C.I. MS (CH₄): 491 (calcd for C₃₂ H₃₅N₅ ·H⁺ : 490); FAB MS (3-nitrobenzyl alcohol matrix, 8 keVacceleration): m/e 512 (calcd for C₃₂ H₃₄ N₅ Na⁺ : 512); IR (KBr)ν=3420, 2960, 2920, 2860, 1600, 1560, 1540, 1370, 1350, 1255, 1210,1080, 1050, 980, 940, 905, 750 cm⁻¹ ; UV/VIS (CHCl₃) λ_(max) nm (ε)327.0 (30,700); 422.5 (60,500); 692.0 (10,100); 752.0 (36,400 ).

Attempts at binding cadmium with ligand 2_(A) were conducted. Severalmilligrams of compound 2_(A) were stirred with an excess of cadmiumchloride in chloroform/methanol as per the direct insertion methodsoutlined above. Even after 2 days, however, UV/VIS (monitoring theQ-type band at 751), indicated that little or no metal insertion hadtaken place. Due to the difficulty of preparing compound or ligand 2_(A)and the obvious success of the direct insertion procedures describedherein, no attempts were made to examine other metallation methods.

The preparation of complex 3_(A) ·Cl was as follows. The sp³ form of theligand (1_(A)) (40 mg, 0.08 mmol) was stirred with cadmium chloride(21.4 mg, 0.08 mmol) in chloroform/methanol (150 ml, v/v 2/1) for 1 day.The dark green reaction mixture was concentrated under reduced pressureon a rotary evaporator and chromatographed through silica gel usingfirst pure chloroform and then chloroform/methanol (v/v 10/1) aseluents. After discarding several leading red bands, the dark green bandwas collected and taken to dryness in vacuo to give compound 3_(A) ·Cl.This material was recrystallized from chloroform/n-hexane to giveanalytical pure compound 3_(A) ·Cl as a dark green powder in 24% yield.For 3_(A) ·Cl: ¹ H-NMR (CDCl₃): δ=1.55-1.67 (12H, m, CH₂ CH₃), 3.03 (6H,s, CH₃), 3.04-3.55 (8H, m, CH₂ CH₃), 8.27 (2H, m, phen. CH), 9.23 (2H,s, CH═N), 9.40 (2H, m, phen. CH), 11.30 (2H, s, CH═C); ¹³ C NMR (CDCl₃):δ=9.8, 17.3, 18.1, 19.1, 19.2, 117.6, 117.8, 128.4, 132.7, 138.2, 139.3,145.4, 146.7, 150.5, 153.5, 155.0; FAB MS (3-nitrobenzyl alcohol matrix,8 keV acceleration): m/e 602 (¹¹⁴ Cd, M⁺, 100), 601 (¹¹³ Cd, M⁺, 64),600 (¹¹² Cd, M⁺, 84); IR (KBr) ν=2950, 2910, 2855, 1635, 1605, 1380,1255, 1210, 1090, 1010, 795 cm⁻¹ ; UV/VIS λ_(max) nm (ε) 327.0 (32,800);424.0 (72,700); 704.5 (11,000); 767.5 (41,200); anal. calcd for C₃₂ H₃₄N₅ Cd·Cl·(1/2·H₂ O): C, 59.54; H, 5.46; N, 10.85. Found: C, 59.78; H,5.32; N, 10.80.

The preparation of complex 3_(A) ·NO₃ was as follows. The sp³ form ofthe ligand (1_(A))¹⁴ (40 mg, 0.08 mmol) was stirred with cadmium nitratetetrahydrate (31 mg, 0.1 mmol) in chloroform/methanol (150 ml, v/v=1/2)for 1 day. The dark green reaction mixture was then concentrated andpurified by chromatography on silica gel as described above. Theresulting crude material was then recrystallized fromchloroform/n-hexane to give analytical pure 3·NO₃ in 27% yield.⁴⁵ For3_(A) ·NO₃ : ¹ H NMR (CDCl₃): δ=1.55-1.70 (12H, m, CH₂ CH₃), 3.04 (6H,s, CH₃), 3.42-3.55 (8H, m, CH₂ CH₃), 8.27 (2H, m, phen. CH), 9.20 (2H,S, CH═N), 9.30 (2H, m, Phen. CH), 11.07 (2H, s, CH═C); FAB MS(3-nitrobenzyl alcohol matrix, 8 KeV acceleration): m/e 602 (¹¹⁴ Cd, M⁺,100), 601 (¹¹³ Cd, M⁺,61), 600 (¹¹² Cd, M⁺, 87): IR (KBr) ν=2960, 2920,2860, 1600, 1550, 1440, 1375, 1200, 1130, 1075, 1040, 975, 930, 900, 740cm⁻¹ ; UV/Vis λ_(max) nm (ε)=328.0 (39,900) 425.0 (82,800), 706.0(14,400), 770 (49,800); Anal. calcd for C₃₂ H₃₄ N₅ Cd·NO₃ ·(1/2H₂ O): C,57.19; H, 5.25; N, 12.50. Found: C, 57.12; H, 5.19; N, 11.80.

Attempts to demetallate complex 3_(A) ·NO₃ were made. In an effort toobtain the free sp² bridged ligand 2_(A), the above complex inchloroform was stirred for several hours in the presence of sodiumsulfide and independently with sodium thiosulfate. No significantchanges in optical properties were observed. Although this did not ruleout the possibility that changes in axial ligation might be takingplace, these observations provided reasonable evidence that little or nodemetallation occurs under the reaction conditions. In the case ofsodium sulfide, this critical conclusion was further supported by FABMS: Other than the starting cationic complex 3_(A), no evidence wasobtained for any moderate to high molecular weight volatile products inthe mass spectrum. When subject to treatment with aqueous acid, complex3_(A) ·NO₃ appears to undergo hydrolysis (at the imine residues) andhence demetallation. The rate of this process, however, is strongly pHdependent, the half-life being, for instance, on the order of severalhours in the presence of ca. 0.1N HCl.

Preparation and isolation of complex 4b_(A) ·NO₃. The sp³ form of theligand (1_(A)) (40 mg, 0.08 mmol) was stirred with cadmium nitratetetrahydrate (31 mg, 0.1 mmol) in chloroform/methanol (150 ml, v/v=1/2)for 1 day. The dark green reaction mixture was concentrated on a rotaryevaporator and chromatographed through Sephadex using first neatchloroform and then chloroform/methanol (v/v 10/1) as eluents. Afterdiscarding several leading red bands, the dark green band was collectedand concentrated to give a dark green solid. This was recrystallizedfrom chloroform/n-hexane to give a mixture of crystalline andnoncrystalline solids in 27% yield. For this bulk material: ¹ H-NMR(CDCl₃): δ=1.55-1.72 (12H, m, CH₂ CH₃), 3.04 (6H, s, CH₃), 3.45-3.58(8H, m, CH₂ CH₃), 6.4 (ca. 3/5H, br. s, BzIm), 6.81(ca. 6/5H, br. s.,BzIm), 7.27 (ca. 6/5H, s, BzIm), 829 (2H, m, phen. CH), 9.21 (2H, s,CH═N), 9.32 (2H, m, phen. CH), 11.08 (2H, s, CH═C); FAB MS(3-nitrobenzyl alcohol matrix, 8 keV acceleration): m/e 602 (¹¹⁴ Cd, M⁺,100) 601 (¹¹³ Cd, M⁺, 67), 600 (¹¹² Cd, M⁺, 78); IR (KBr) ν=2970, 2935,2875, 1560, 1382, 1356, 1300, 1258, 1212, 1085, 1050, 985, 910, 755 cm⁻¹; uv/vis λ_(max) nm (ε) 325.0 (29,000); 425.0 (64,400); 710.5 (9,800);767.5 (38,500); anal Found: C, 42.42; H, 4.28; N, 10.34 (calcd for C₃₂H₃₄ N₅ Cd·NO₃ ·(1/2H₂ O): C, 57.19; H, 5.25; N, 12.50; calcd for C₃₂ H₃₄N₅ Cd·NO₃ ·BzIm·CHCl₃ : C, 53.35; H, 4.59; N, 12.44; calcd for C₃₂ H₃₄N₅ Cd·NO₃ ·CHCl₃ : C, 41.26; H, 3.66; N, 8.25). The single crystal of4b_(A) used for the X-ray structure determination was isolated fromresidual noncrystalline material following a second recrystallizationwhich involved layering a concentrated solution of the above crudematerial in CDCl₃ with n-hexane and letting stand for several months inthe refrigerator.

Preparation of complex 5a_(A) ·NO₃. In a fashion similar to that used toprepare the crude cadmium-containing complex described above, the sp³form of the ligand (1_(A)) was treated with cadmium nitrate tetrahydrateand purified on Sephadex. To a ca. 0.7 ml, 0.005M sample of this productin CDCl₃, was added 25 μl of pyr-D₅. The resulting solution was layeredwith n-hexane, and placed in the refrigerator. After several months,green crystals were isolated in nearly quantitative yield. The molecularcomposition of these crystals was determined, on the basis of the singlecrystal X-ray diffraction analysis reported earlier,¹¹ to be 5a·NO₃·CHCl₃. NMR (CDCl₃ /pyr-D₅) δ=1.55-1.70 (12H, m, CH₂ CH₃), 3.22 (3H, s,CH₃), 3.45-3.56 (8H, m, CH₂ CH₃), 8.40 (2H, m, phen. CH), 9.32 (2H, s,CH═N), 9.75 (2H, m, phen. CH), 11.62 (2H, s, CH═C); UV/VIS (CHCl₃ -pyrv/v 10/1) λ_(max) nm (ε) 321.5 (45,000), 426.5 (79,000), 700.5 (13,500),765.5 (51,900).

The ¹ H NMR titration of 3_(A) ·NO₃ with BzIm or pyr-D₅ was done.Rigorously purified complex 3_(A) ·NO₃ was dried at 80° C. under reducedpressure (1 mmHg) for 1 day. Starting samples for titration were thenprepared by dissolving this five coordinate complex (3.32 mg, 0.005mmol) in 0.7 to 0.75 ml of CDCl₃ and transferring quantitatively to anNMR tube. To such samples were then added increasing aliquots of eitherBzIm or pyr-D₅ (as solutions of known concentration in CDCl₃) andrecording the chemical shift of the "meso" protons at 27° C. Controlexperiments were also carried out by adding known quantities of CF₃ CO₂H, D₂ O, and NH₄ NO to similar stock solutions of 3·NO₃. In thesevarious ¹ H NMR titrations the absolute chemical shifts for any givenbase to ligand ratio were found to vary by less than 0.05 ppm betweenindependent runs, with the values of δ-δ_(o), the critical observableused for the K_(eq). determinations (see below), being found to varyeven less (generally ≦0.003 ppm).

Determination of Binding Constants. Inspection of FIG. 14 and FIG. 15show that the binding of BzIm to cation 3_(A) may be considered as twowell-separated equilibrium processes. The chemical shift data obtainedfor the "meso" signals as a function of added BzIm were thus analyzed assuch at both very low and very high conversion: Standard Scatchard(single reciprocal) plots³⁸ were constructed by plotting(δ-δ_(o))/[BzIm] vs (δ-δ_(o)) according to equation 4 (which correspondsto eq. 5.13 of ref. 38), obtaining K as the absolute value of the slopeand the term (δ.sub.∞ -δ_(o))K as the intercept.

    (δ-δ.sub.o)/[BzIm]=-K(δ-δ.sub.o)+(δ.sub..infin. -δ.sub.o)K                                        (4)

Here δ is the observed chemical shift, δ_(o) is the initial chemicalshift of the pure five or six coordinate starting complex (3_(A) ·NO₃ or4b_(A) ·NO₃), δ.sub.∞ the chemical shift calculated for the final mono-or bisligated complexes 4b_(A) ·NO₃ or 5a_(A) ·NO₃, K the equilibriumconstant in question, and [BzIm] the concentration of is, uncomplexedbenzimidazole. In both the low and high conversion regimes, it provednecessary to correct for bound benzimidazole so as to obtain validexpressions for [Bzim] in terms of added benzimidazole ([BzIm]). Thiswas done in a straightforward manner according to the expressions givenin equations 5 and 6, where [lig]_(o) represents the concentration ofthe starting five coordinate ligand 3_(A) ·NO₃.

    [BzIm]=[BzIm].sub.o -[lig].sub.o (δ-δ.sub.o)/δ.sub.∞ -δ.sub.o) at low [BzIm].sub.o                                              (5)

    [BzIm]≅[BzIm].sub.o -[lig].sub.o at high [BzIm].sub.o(6)

Using these corrected values for [BzIm], straight line Scatchard plotswere obtained with R≧0.99 and 0.98 respectively or the low and high[Bzim]_(o) regimes giving values of K₁ and K₂ of 1.80×10⁴ M⁻¹ and12.9M⁻¹ respectively (see supplementary material). The value for K₁ isconsidered to be quite reliable (estimated error≦15%); the lowsolubility of BzIm and the resulting incomplete nature of the titrationassociated with the formation of 5a_(A) ·NO₃, however, makes the valueobtained for K₂ somewhat more approximate (estimated error≦25%).⁴⁹

The changes in "meso" proton chemical shift as a function of added [pyr]shown in FIG. 16 indicate a clear absence of two distinct bindingregimes. Moreover, as expected, attempts to fit the data as a simplemonoligation process (to give 6CN material) according to eq. 1 did notwork. It therefore proved necessary to analyze the data in terms of twoconcurrent equilibrium processes. This was done using the convenientiterative procedure outlined by Connors.³⁸ Here the equations ofinterest, corresponding to eqs. 4.31 and 4.32 of Connors,³⁸ as adoptedfor NMR analyses, are:

    1/[pyr]-K.sub.1 Δ.sub.11 /(δ-δ.sub.o)=K.sub.1 K.sub.2 [pyr]{Δ.sub.12 /(δ-δ.sub.o)-1}-K.sub.1  (7)

    (δ-δ.sub.o){1+K.sub.1 [pyr]+K.sub.1 K.sub.2 [pyr].sup.2 }/[pyr]=K.sub.1 K.sub.2 Δ.sub.12 [pyr]+K.sub.1 Δ.sub.11(8)

where δ is the observed chemical shift, δ_(o) is the initial chemicalshift of the pure five coordinate starting complex 3·NO₃, Δ₁₁ is thetotal chemical shift difference corresponding to the formation of thepure putative monoligated six coordinate species, Δ₁₂ is the totalchemical shift corresponding to the formation of the bisligated cationicspecies 5a from the initial five coordinate material, and [pyr] is thefree pyridine concentration. A precise expression for [pyr] is given byequation 9,³⁸ where [pyr]_(o) is the concentration total added pyridine,and [lig]_(o) represents the concentration of the starting fivecoordinate ligand 3_(A) ·NO₃.

    [pyr].sub.o =[pyr]+[lig].sub.o (K.sub.1 [pyr]+2K.sub.1 K.sub.2 [pyr].sup.2)/(1+K.sub.1 [pyr]+K.sub.1 K.sub.2 [pyr].sup.2)(9)

Inspection of the binding isotherm (FIG. 16), however, suggested thatthe approximation [pyr]≅[pyr]_(o) would be reasonably valid over much ofthe titration range. Initial iterative solutions of eqs. 7 (plotting1/[pyr]-K₁ Δ₁₁ /(δ-δ_(o)) vs. [pyr]{Δ₁₂ /δ-δ_(o))-1}, giving K₁ K₂ and-K₁ as the slope and intercept respectively) and 8 (plottingδ-δ_(o)){1+K₁ [pyr]+K₁ K₂ [pyr]² }/[pyr] vs. [pyr], giving K₁ K₂ Δ₁₂ andK₁ Δ₁₁ as the slope and intercept respectively) were therefore madeusing this greatly simplifying assumption. They converged quickly togive initial, uncorrected values of K₁ ≅1.5M⁻¹ and K₁ K₂ =308M⁻². Thesevalues confirmed that under the conditions of the experiment (where[3_(A) · NO₃ ]=0.005M), the approximation [pyr]≅[pyr]_(o) is valid towithin ≦4% in the regime of greatest interest, namely3<[pyridine]/[ligand]<10 and 0.005M in 3_(A) ·NO₃. When corrections aremade for this small percentage, final values of K₁ of ≅1.6M⁻¹ and K₁ K₂=315M⁻² are obtained (see supplementary material). We consider itimportant to stress that although the value of K₁ K₂ is well determined(estimated error ≦10%), the nature of the data does not allow K₁ (andhence K₂) to be defined with precision (estimated error≅50%). Thisuncertainty, however, does not detract from the central conclusionsdescribed herein.

X-ray Experimental for complex 4b_(A). For 4b_(A) ·NO₃ ·CHCl₃ : C₄₀ H₄₁N₈ O₃ Cl₃ Cd, M=900.57. The data crystal was a very dark green plate ofdimensions 0.06×0.22×0.44 mm, which was grown by slow diffusion fromCHCl₃ -hexanes and separated from the accompanying noncrystallinematerial as described above. The data were collected on a Nicolet R3diffractometer, with a graphite monochromator, using Mo Kα radiation(λ=0.71069 Å) and a Nicolet LT2 low-temperature delivery system (163°K.). Lattice parameters were obtained from least-squares refinement of26 reflections with 19.2°<2θ<24.4°. The space group was triclinic, P1(No. 2), with Z=2, F(000)=920, a=11.276(4), b=12.845(3), c=14.913(4) Å,α=84.82(2), β=69.57(2), λ=85.84(2)°, v=2014(1) Å, ρ_(C) =1.48 g-cm⁻³.Data were collected using the omega scan technique (7191 reflections,6566 unique, R_(int) = 0.064), 2θ range 4.0°-50.0°, 1.2° ω scan at3°-6°/min. (h=0→14, k=-15, l=-18→18). Four reflections (-2,2,0; 3,2,3;2,-3,-1; -1,0,-4) were remeasured every 146 reflections to monitorinstrument and crystal stability. Decay correction range on I was0.9863-1.076. Data also corrected for L_(P) effects and absorption(based on crystal shape; transmission factor range 0.8533-0.9557,μ=7.867 cm⁻¹). Reflections having F_(o) <6σ (F_(o)) consideredunobserved (3272 reflections). The structure was solved by heavy atomand Fourier methods and refined by full-matrix least-squares proceduresin blocks of 253 and 287 with-anisotropic thermal parameters for thenon-H atoms (except 03A of the disordered NO₃ -- group and the terminalC atoms of the disordered ethyl groups of one pyrrole ring, C29 [siteoccupancy factor 0.44(2)], C29A, C31 [site occupancy factor 0.37(2)] andC31A). Nitrate disordered about two orientations of the N atom (N1B)with site occupancy factors for the minor orientation (O atoms labeledwith A) of 0.45(2). H atoms were calculated and refined with isotropicthermal parameters riding on the relevant C atom. CHCL₃ solvent isdisordered by rotation about a C-Cl bonding axis (ClC-Cll) with siteoccupancy factor for the minor component (Cl atoms labelled with A) of0.43(2). Due to the disorder, the chloroform H atom position was notcalculated. Σw(|F_(o) |-|F_(c) |)² minimized, where w=1/[(σ(F_(o)))²+0.0118(F²)] and σ(F_(o))=0.5 kI^(-1/2) (σ(I)). Intensity, I, given by(^(I) peak^(-I) background)×(scan rate) and k is the correction due toLp effects, absorption and decay. Sigma(I) estimated from countingstatistics; σ(I)=[.sup.(I peak^(+I) background)^(1/2) ×(scan rate)].Final R=0.0781 for 3294 reflections, wR=0.114 (R_(all) =0.143, wR_(all)=0.176) and a goodness of fit=1.00. Maximum |Δ/σ|<0.1 in the finalrefinement cycle and the minimum and maximum peaks in the final ΔF mapwere -0.97 and 1.69 e-/Å³ respectively (in the region of the Cd atom).Data reduction, structure solution and initial refinement wereaccomplished using Nicolet's SHELXTL-PLUS⁵⁰ software package. The finalrefinement was done using SHELX76.⁵¹ Neutral atom scattering factors forthe non-H atoms from Cromer and Mann,⁵² with anomalous-dispersioncorrections from Cromer and Liberman,⁵³ while scattering factors for theH atoms from Stewart, Davidson and Simpson;⁵⁴ linear absorptioncoefficient from the International Tables for X-ray Crystallography(1974).⁵⁵ The least-squares planes program was supplied by Cordes;⁵⁶other computer programs from reference 11 of Gadol and Davis.⁵⁷

Table 1 shows sectional coordinate or equivalent isotropic thermalparameters (A²) for non-hydrogen atoms of 4b_(A) ·CHCl₃. Table 2 showsbond lengths (Å) and angles (°) for non-hydrogen atoms of cation 4b_(A).TABLE 1. Fractional coordinates and isotropic or equivalentisotropic^(a) thermal parameters (Å²) for non-hydrogen atoms of 4b_(A)·NO₃ ·CHCl₃.

    ______________________________________                                        Atom  x          y          z        U                                        ______________________________________                                        Cd    .37392(8)  .12411(6)  .04542(7)                                                                              .0573(4)                                 N1    .5259(9)   .1813(7)   .1140(9) .065(5)                                  C2    .5321(13)  .1503(10)  .2032(11)                                                                              .062(6)                                  C3    .6213(12)  .2115(11)  .2219(10)                                                                              .064(6)                                  C4    .6661(11)  .2783(9)   .1439(11)                                                                              .061(6)                                  C5    .6066(12)  .2575(9)   .0765(11)                                                                              .060(6)                                  C6    .6340(12)  .3124(9)   -.0106(12)                                                                             .065(7)                                  C7    .5828(11   .3039(8)   -.0848(11)                                                                             .063(6)                                  N8    .4929(9)   .2314(7)   -.0754(8)                                                                              .056(4)                                  C9    .4750(12)  .2437(10)  -.1581(11)                                                                             .070(6)                                  C10   .5492(14)  .3233(11)  -.2225(10)                                                                             .075(6)                                  C11   .6178(12)  .3604(9)   -.1782(11)                                                                             .069(6)                                  C12   .3827(14)  .1784(12)  -.1763(10)                                                                             .073(6)                                  N13   .3236(10)  .1169(8)   -.1068(8)                                                                              .057(4)                                  C14   .2359(11)  .0438(11)  -.1074(10)                                                                             .063(6)                                  C15   .1851(13)  .0468(12)  -.1834(10)                                                                             .072(6)                                  C16   .1028(14)  -.0250(12) -.1825(10)                                                                             .067(6)                                  C17   .0648(13)  -.0982(12) -.1067(11)                                                                             .071(7)                                  C18   .1077(12)  -.1040(9)  -.0331(11)                                                                             .074(7)                                  C19   .1952(12)  -.0304(8)  -.0304(11)                                                                             .064(6)                                  N20   .2428(10)  -.0258(9)  .0420(9) .065(5)                                  C21   .2112(13)  -.0873(9)  .1201(11)                                                                              .065(6)                                  C22   .2643(12)  -.0711(9)  .1918(11)                                                                              .065(7)                                  N23   .3510(10)  .0013(6)   .1716(9) .061(5)                                  C24   .3782(14)  .0029(10)  .2535(13)                                                                              .076(7)                                  C25   .3076(14)  -.0727(10) .3260(12)                                                                              .080(7)                                  C26   .2349(15)  -.1183(9)  .2854(11)                                                                              .073(7)                                  C27   .4669(13)  .0715(10)  .2649(11)                                                                              .067(6)                                  C28   .6508(14)  .2018(13)  .3139(11)                                                                              .084(7)                                  C29   .553(5)    .234(4)    .400(3)  .105(15)                                 C29A  .590(3)    .282(2)    .381(2)  .082(8)                                  C30   .765(2)    .3611(12)  .1304(13)                                                                              .088(8)                                  C31A  .710(3)    .450(2)    .194(2)  .101(9)                                  C31   .704(3)    .475(2)    .134(2)  .040(7)                                  C32   .7108(13)  .4478(10)  -.2122(11)                                                                             .072(6)                                  C33   .6487(14)  .5579(11)  -.1933(13)                                                                             .088(8)                                  C34   .553(2)    .3512(14)  -.3236(10)                                                                             .096(8)                                  C35   .132(2)    -.1983(11) .3305(13)                                                                              .093(8)                                  C36   .305(2)    -.0887(12) .4286(11)                                                                              .084(8)                                  C37   .229(2)    -.007(2)   .4904(14)                                                                              .125(11)                                 N1A   .2036(10)  .2375(6)   .1132(8) .059(5)                                  C2A   .1817(13)  .3278(11)  .060(2)  .095(9)                                  N3A   .0853(13)  .3834(8)   .1207(14)                                                                              .101(8)                                  C4A   .0536(12)  .3373(11)  .2074(14)                                                                              .073(7)                                  C5A   -.043(2)   .3615(13)  .300(2)  .103(11)                                 C6A   -.056(2)   .296(2)    .378(2)  .116(12)                                 C7A   .0178(15)  .1969(15)  .3762(10)                                                                              .089(7)                                  C8A   .1056(14)  .1750(12)  .2886(11)                                                                              .070(7)                                  C9A   .1212(11)  .2418(9)   .2079(10)                                                                              .053(5)                                  C11   .3144(6)   .4520(4)   .5507(4) .129(3)                                  C12   .3058(12)  .2261(8)   .5862(6) .118(5)                                  C13   .1613(13)  .3391(13)  .4877(8) .165(8)                                  C12A  .056(2)    .4327(14)  .5339(13)                                                                              .155(10)                                 C13A  .2064(15)  .2450(12)  .5529(14)                                                                              .157(9)                                  C1C   .198(3)    .357(2)    .5834(15)                                                                              .152(15)                                 N1B   .9690(14)  .6403(9)   .1553(10)                                                                              .073(6)                                  O1    .985(3)    .588(2)    .079(2)  .072(11)                                 O2    .943(4)    .734(2)    .167(2)  .17(2)                                   O3    .958(3)    .587(2)    .233(2)  .123(14)                                 O1A   .853(3)    .660(4)    .199(3)  .19(2)                                   O2A   1.043(3)   .598(3)    .089(3)  .065(13)                                 O3A   1.030(5)   .676(4)    .173(3)  .16(2)                                   ______________________________________                                         .sup.a For anisotropic atoms, the U value is U.sub.eq, calculated as          U.sub.eq = 1/3 Σ.sub.i Σ.sub.j U.sub.ij a.sub.i *a.sub.j          *A.sub.ij where A.sub. ij is the dot product of the i.sup.th and j.sup.th     direct space unit cell vectors.                                          

                  TABLE 2                                                         ______________________________________                                        Bond Lengths (Å) and Angles (°)                                    for non-H atoms of cation 4b.sub.A.                                           1         2        3        1-2   1-2-3                                       ______________________________________                                        C2        N1       C5       1.38(2)                                                                             107.2(13)                                   C5        N1                1.33(2)                                           C3        C2       C27      1.43(2)                                                                             124.(2)                                     C3        C2       N1             109.5(11)                                   C27       C2       N1       1.37(2)                                                                             126.3(15)                                   C4        C3       C28      1.35(2)                                                                             129.3(14)                                   C4        C3       C2             105.9(14)                                   C28       C3       C2       1.51(2)                                                                             124.7(12)                                   C5        C4       C30      1.44(3)                                                                             126.9(13)                                   C5        C4       C3             107.8(12)                                   C30       C4       C3       1.55(2)                                                                             125.(2)                                     C6        C5       N1       1.37(2)                                                                             129.(2)                                     C6        C5       C4             121.7(12)                                   N1        C5       C4             109.5(13)                                   C7        C6       C5       1.43(3)                                                                             129.2(12)                                   N8        C7       C11      1.39(2)                                                                             110.7(14)                                   N8        C7       C6             121.3(12)                                   C11       C7       C6       1.45(2)                                                                             127.8(12)                                   C9        N8       C7       1.31(2)                                                                             103.3(11)                                   C10       C9       C12      1.43(2)                                                                             127.(2)                                     C10       C9       N8             113.6(14)                                   C12       C9       N8       1.49(2)                                                                             119.7(11)                                   C11       C10      C34      1.32(2)                                                                             127.8(13)                                   C11       C10      C9             106.7(14)                                   C34       C10      C9       1.51(2)                                                                             125.(2)                                     C32       C11      C7       1.52(2)                                                                             125.(2)                                     C32       C11      C10            129.1(15)                                   C7        C11      C10            105.7(11)                                   N13       C12      C9       1.26(2)                                                                             115.2(15)                                   C14       N13      C12      1.41(2)                                                                             126.1(14)                                   C15       C14      C19      1.43(2)                                                                             119.5(13)                                   C15       C14      N13            121.7(12)                                   C19       C14      N13      1.39(2)                                                                             118.7(14)                                   C16       C15      C14      1.25(2)                                                                             120.2(13)                                   C17       C16      C15      1.37(2)                                                                             119.(2)                                     C18       C17      C16      1.34(3)                                                                             123.(2)                                     C19       C18      C17      1.43(2)                                                                             120.0(13)                                   N20       C19      C14      1.37(2)                                                                             116.6(12)                                   N20       C19      C18            125.7(12)                                   C14       C19      C18            118.(2)                                     C21       N20      C19      1.30(2)                                                                             124.2(13                                    C22       C21      N20      1.43(3)                                                                             118.3(13)                                   N23       C22      C26      1.34(2)                                                                             113.(2)                                     N23       C22      C21            118.0(13)                                   C26       C22      C21      1.41(2)                                                                             129.1(12)                                   C24       N23      C22      1.36(2)                                                                             104.7(12)                                   C25       C24      C27      1.44(2)                                                                             125.(2)                                     C25       C24      N23            111.1(14)                                   C27       C24      N23      1.44(2)                                                                             123.9(12)                                   C26       C25      C36      1.37(3)                                                                             127.9(13)                                   C26       C25      C24            105.(2)                                     C36       C25      C24      1.52(3)                                                                             127.(2)                                     C35       C26      C22      1.54(2)                                                                             124.(2)                                     C35       C26      C25            129.(2)                                     C22       C26      C25            106.3(12)                                   C2        C27      C24            130.(2)                                     C29       C28      C3       1.44(4)                                                                             118.(3)                                     C29A      C28      C3       1.46(3)                                                                             115.(2)                                     C31A      C30      C4       1.51(3)                                                                             112.(2)                                     C31       C30      C4       1.57(3)                                                                             111.(2)                                     C33       C32      C11      1.54(2)                                                                             114.0(11)                                   C37       C36      C25      1.47(2)                                                                             114.(2)                                     C9A       N1A      C2A      1.40(2)                                                                             108.5(10)                                   C2A       N1A               1.40(2)                                           N3A       C2A      N1A      1.35(2)                                                                             107.(2)                                     C4A       N3A      C2A      1.31(3)                                                                             109.4(14)                                   C5A       C4A      C9A      1.47(3)                                                                             115.(2)                                     C5A       C4A      N3A            133.3(14)                                   C9A       C4A      N3A      1.40(2)                                                                             111.2(13)                                   C6A       C5A      C4A      1.34(3)                                                                             120.(2)                                     C7A       C6A      C5A      1.47(3)                                                                             124.(2)                                     C8A       C7A      C6A      1.37(2)                                                                             115.(2)                                     C9A       C8A      C7A      1.38(2)                                                                             121.7(14)                                   N1A       C9A      C4A            103.7(12)                                   N1A       C9A      C8A            132.1(11)                                   C4A       C9A      C8A            124.1(13)                                   ______________________________________                                    

The characterization by X-ray diffraction analysis of a six coordinatepentagonal pyramidal cadmium(II) cationic complex 4b_(A) derived from anovel aromatic 22 π-electron pentadentate "expanded porphyrin" ligand(2_(A)) is described. The X-ray structure reveals the five central donoratoms of the macrocycle to be coordinated to the cadmium(II) cationwhich in turn lies 0.334(2) Å above the mean plane of the macrocyle andis further ligated by an apical benzimidazole ligand. As is true in thecorresponding pentagonal bipyramidal bispyridine adduct 5a_(A), theX-ray structure of cation 4b_(A) indicates the macrocyclic ligand to benearly planar (maximum deviation, 0.154(13) Å for C15) with the fivedonor nitrogen atoms defining a near circular cavity with acenter-to-nitrogen radius of ≅2.42 Å. The crystals of 4b_(A) ·NO₃ usedfor the X-ray diffraction analysis were isolated from an inhomogeneousmixture of crystalline and noncrystalline material obtained followingtreatment of the sp³ form of the ligand (1_(A)) with Cd(NO₃)₂ ·(H₂ O)₄and subsequent purification on sephadex. The proton NMR spectrum inCDCl₃ of this bulk material is essentially identical to that of the purefive coordinate complex 3_(A) prepared independently, but showed thepresence of a broad feature at ca. 6.4 ppm and two sharper peaks at 6.81and 7.27 ppm ascribable to the bound benzimidazole ligand. Thesediagnostic ligand features are reproduced upon titrating the pure fivecoordinate complex 3_(A) with roughly 3/5 equivalent of benzimidazole.This finding suggests that the bulk material from which crystals of4b_(A) ·NO₃ were isolated consists of a mixture of crystalline andnoncrystalline six and five coordinate species and supports thehypothesis that the bound benzimidazole found in cation 4b_(A) isderived from degradative side reactions associated with the metalinsertion and accompanying ligand oxidation. From these titrations thevalues for the sequential formation constants (K₁ and K₂) for thebinding of the first and second equivalents of benzimidazole to the fivecoordinate cationic complex 3_(A) were determined to be 1.8×10⁴ M⁻¹respectively. For the complexation of pyridine to 3_(A) ·NO₃, K₁ ·K₂values of 1.6M⁻¹ and 315M⁻² respectively were determined from similar ¹H NMR titrations. These results indicate that inbenzimidazole-containing chloroform solutions an extended concentrationrange exists wherein the pentagonal pyramidal complex 4b_(A) is theprimary cadmium containing species, whereas in the presence of pyridineit is either the unligated complex 3_(A) or the coordinatively saturatedpentagonal bipyramidal species 5a_(A) which will dominate in solution.

Published literature references in the following list are incorporatedby reference herein for the reasons cited.

REFERENCES

1. "The Porphyrins"; Dolphin, D., Ed.; Academic Press: New York,1978-1979; Vols. I-VII.

2. (a) Day, V. W.; Marks, T. J.; Wachter, W. A. J. Am. Chem. Soc. 1975,97, 4519-4527. (b) Marks, T. J.; Stojakovic, D. R. J. Am. Chem. Soc.1978, 100, 1695-1705. (c) Cuellar, E. A.; Marks, T. J. Inorg. Chem.1981, 20, 3766-3770.

3. Bauer, V. J.; Clive, D. R.; Dolphin, D.; Paine, J. B. III; Harris,.F. L.; King, M. M.; Loder, J.; Wang, S.-W. C.; Woodward, R. B. J. Am.Chem. Soc. 1983, 105, 6429-6436. To date only tetracoordinated metalcomplexes have been prepared from these potentially pentadentateligands.

4. Broadhurst, M. J.; Grigg, R.; Johnson, A. W. J. Chem. Soc. PerkinTrans. 1, 1972, 2111-2116.

5. (a) Broadhurst, M. J.; Grigg, R.; Johnson, A. W. J. Chem. Soc., Chem.Commun. 1969, 23-24; Broadhurst, M. J.; Grigg, R.; Johnson, A. W. J.Chem. Soc., Chem. Commun. 1969, 1480-1482; Broadhurst, M. J.; Grigg, R.;Johnson, A. W. J. Chem. Soc., Chem. Commun.. 1970, 807-809.

6. (a) Berger, R. A.; LeGoff, E. Tetrahedron Lett. 1978, 4225-4228. (b)LeGoff, E.; Weaver, O. G. J. Org. Chem. 1987, 710-711.

7. (a) Rexhausen, H.; Gossauer, A. J. Chem. Soc., Chem. Commun. 1983,275. (b) Gossauer, A. Bull. Soc. Chim. Belg. 1983, 92, 793-795.

8. Gosmann, M.; Franck, B. Angew. Chem. 1986, 98, 1107-1108; Angew.Chem. Int. Ed. Eng. 1986, 25, 1100-1101.

9. For examples of a porphyrin-like systems with smaller centralcavities see: (a) Vogel, E.; Kocher, M.; Schmickler, H.; Lex, J. Angew.Chem. 1986, 98, 262-263; Angew. Chem. Int. Ed. Eng. 1986, 25, 257-258.(b) Vogel, E.; Balci, M.; Pramod, K.; Koch, P.; Lex. J. Ermer, O. Angew.Chem. 1987, 99, 909-912; Angew. Chem. Int. Ed. Eng. 1987, 26, 928-931.

10. For examples of large nonaromatic pyrrole-containing macrocyclessee: (a) Acholla, F. V.; Mertes, K. B. Tetrahedron Lett. 1984,3269-3270. (b) Acholla, F. V.; Takusagawa, F.; Mertes, K. B. J. Am.Chem. Soc. 1985, 6902-6908. (c) Adams, H.; Bailey, N. A.; Fenton, D. A.;Moss, S.; Rodriguez de Barbarin, C. O.; Jones, G. J. Chem. Soc., DaltonTrans. 1986, 693-699. (d) Fenton, D. E.; Moody, R. J. Chem. Soc., DaltonTrans. 1987, 219-220.

11. Sessler, J. L.; Murai, T.; Lynch, V.; Cyr, M. J. Am. Chem. Soc.1988, 110, 5586-5588.

12. Sessler, J. L.; Cyr, M.; Murai, T. Comm. Inorg. Chem., in press.

13. Stark, W. M.; Baker, M. G.; Raithby, P. R.; Leeper, F. J.;Battersby, A. R. J. Chem. Soc., Chem. Commun. 1985, 1294.

14. Sessler, J. L.; Johnson, M. R.; Lynch, V. J. Org. Chem. 1987, 52,4394-4397.

15. Sessler, J. L.; Johnson, M. R.; Lynch, V.; Murai, T. J. Coord.Chem., in press.

16. Sessler, J. L.; Murai, T. Tetrahedron Lett., to be submitted.

17. Hoard, J. L. In Porphyrins & Metalloporphyrins; Chapter 8, Smith,K., Ecl.; Elsevien, Amsterdam, 1975.

18. Chemical & Engineering News Aug. 8, 1988, 26-27.

19. For reviews see: (a) Drew, M. G. B. Prog. Inorg. Chem. 1977, 23,67-210. (b) Melson, G. A. in "Coordination Chemistry of MacrocyclicCompounds", Melson, G. A., Ed.; Plenum: New York, 1979, Chapter 1. (c)N. F. Curtis, in "Coordination Chemistry of Macrocyclic Compounds",Melson, G. A., Ed.; Plenum: New York, 1979, Chapter 4. (d) Nelson, S. M.Pure and Appl. Chem. 1980, 52, 2461-2476. (e) Lindoy, L. F. in"Synthesis of Macrocycles", Izatt, R. M. and Christensen, J. J., Eds.;J. Wiley: New York, 1987, Chapter 2. (f) Newkome, G. R.; Gupta, V. K.;Sauer, J. D. in "Heterocyclic Chemistry", Newkome, G. R., Ed.; J. Wiley:New York, 1984, Vol. 14, Chapter 3. (g) De Sousa, M.; Rest, A. J. Adv.Inorg. Chem. Radiochem. 1978, 21, 1-40. (h) See also ref. 12.

20. For recent examples of bipyridine-derived systems and relatedpentadentate ligands, see: (a) Ansell, C. W. G.; Lewis, J.; Raithby, P.R.; Ramsden, J. N.; Schroder, M. J. Chem. Soc., Chem. Commun., 1982,546-547. (b) Lewis, J.; O'Donoghue, T. D.; Raithby, P. R. J. Chem. Soc.,Dalton Trans., 1980, 1383-1389. (c) Constable, E. C.; Chung, L.-Y.;Lewis, J.; Raithby, P. R. J. Chem. Soc., Chem. Commun., 1986, 1719-1720.(d) Constable, E. C.; Holmes, J; M.; McQueen, R. C. S. J. Chem. Soc.,Dalton Trans., 1987, 5-8.

21. Ochai, E.-I. "Bioinorganic Chemistry", Allyn and Bacon: Boston,1977, pp. 475-476.

22. Klaasen, C. D. in "The Pharmacological Basis of Therapeutics, 6thEdition", Gilman A. G.; Goodman, L. S.; Gilman, A., Eds., Macmillan: NewYork, 1980 Chapter 69, pp. 1632-1633.

23. For recent reviews see: (a) Summers, M. F. Coord. Chem. Rev. 1988,86, 43-134. (b) Ellis, P. D. Science 1983, 221, 1141-1146. (c) Ellis, P.D. in "The Multinuclear Approach to NMR Spectroscopy", Lambert, J. B.;Riddell, F. G., Eds.; D. Reidel: Amsterdam, 1983, pp. 457-523.

24. Interestingly, pentagonal pyramidal and pentagonal bipyramidalgeometries have been observed in two very closely related pentadentatemacrocyclic Schiff base ligands which differ only in the size of thering (16 vs. 17 atoms); see: (a) Nelson, S. M.; McFall, S. G.; Drew, M.G. B.; Othman, A. H. J. Chem. Soc., Chem. Commun. 1977, 167-168, and (b)Drew, M. G. B.; McFall, S. G.; Nelson, S. M. J. Chem. Soc., DaltonTrans. 1977, 575-581.

25. OEP=octaethylporphyrin, TPP=tetraphenylporphyrin, andPPIXDME=protoporphyrin IX dimethyl ester, with the prefixes H₂ and Cdreferring to the free-base and cadmium(II) forms respectively;BzIm=benzimidazole; pyr=pyridine.

26. Rodesiler, P. F.; Griffith, E. H.; Ellis, P. D.; Amma., E. L. J.Chem. Soc., Chem. Commun., 1980, 492-493.

27. Hazell, A. Acta Cryst. 1986, C42, 296-299.

28. "Texaphyrin" 2 and its derivatives can be formulated as either abenzannelated [18]annulene as an overall 22 π-electron aromatic system.On the basis of preliminary molecular orbital calculations, and spectralcomparisons to an 18 π-electron macrocyclic analogue of 3·NO₃ derivedfrom diaminomalionitrile, for which a lowest energy Q-type transition of692 nm is observed, we currently favor the 22 π-electron formulation:Hemmi, G.; Krull, K., Cyr, M., Sessler, J. L., unpublished results.

29. Drago, R. S. "Physical Methods in Chemistry", W. B. Saunders:Philadelphia, 1977, Chapter 5.

30. Miller, J. R.; Dorough, G. D. J. Am. Chem. Soc. 1952, 74, 3977-3981.

31. Kirksey, C. H.; Hambright, P. Inorg. Chem. 1970, 9, 958-960.

32. For general discussions see: Gouterman, M. In ref. 1, Vol. III,Chapter 1.

33. Dorough, G. D.; Miller, J. R. J. Am. Chem. Soc. 1951, 73, 4315-4320.

34. Edwards, L.; Dolphin, D. H.; Gouterman, M.; Adler, A. D. J. Mol.Spectroscopy, 1971, 38, 16-32.

35. Johnson, M. R.; Cyr, M.; Sessler, J. L., unpublished results.

36. (a) Scheer, H.; Katz, J. J. In ref. 17, Chapter 10. (b) Janson, T.R.; Katz, J. J. In ref. 1, Vol IV, Chapter 1.

37. "Aldrich Library of NMR Spectroscopy, 2nd ed.", Pouchert, C. J.,Ed., Aldrich Chemical Co.: Milwaukee, 1983; Vol. 2, p. 558.

38. Connors, K. A. "Binding Constants", J. Wiley: New York, 1987.

39. We ascribe much of this stability to kinetic factors: As detailedherein, insertion of Cd²⁺ into the preformed "texaphyrin" 2 did not takeplace at an appreciable rate. This suggests that the kinetic barrier issubstantial for metal insertion; the same is likely to be true fordecomplexation.

40. The addition of traces of acid causes the "meso" signals to shiftdramatically to higher field, moving, for instance, by 0.113 ppm afterthe addition of 1 equivalent of CF₃ CO₂ H; this suggests that thequantitative K_(eq). titration experiments are in fact reflecting basebinding to cadmium and not simple deprotonation of an adventitiouslyprotonated metal complex.

41. For general discussions see: (a) Ellis, P. E., Jr.; Linard, J. E.;Szymanski, T.; Jones, R. D.; Budge, J. R.; Basolo, F. J. Am. Chem. Soc.1980, 102, 1889-1896. (b) Brault, D.; Rougeee, M. Biochemistry, 1975,13, 4591-4597. (c) Collman, J. P.; Brauman, J. I.; Doxsee, K. M.;Halbert, T. R.; Bunnenberg, E.; Linder, R. E.; LaMar, G. N.; Del Gaudio,J.; Lang, G.; Spartalian, K. J. Am. Chem. Soc. 1980, 102, 4182-4192. (d)Traylor, T. G. Acc. Chem. Res. 1981, 14, 102-109.

42. (a) Collman, J. P.; Brauman, J. I.; Doxsee, K. M.; Sessler, J. L.;Morris, R. M.; Gibson, Q. H. Inorg. Chem. 1983, 22, 1427-1432.

43. See for instance: (a) Collman, J. P.; Reed, C. A. J. Am. Chem. Soc.1973, 95, 2048-2049. (b) Wagner, G. C.; Kassner, R. J. Biochim. Biophys.Acta 1975, 392, 319-327. (c) See also refs. 41b-41d.

44. Preliminary photochemical studies indicate that followingphotoexcitation at 350 nm, the excited triplet of cation 3 is formed inroughly 80% quantum yield. In the absence of oxygen, the observedtriplet lifetime is 54 μs; in the presence of air, the triplet state isquenched completely by formation of singlet oxygen: Mallouk, T.;Sessler, J. L., unpublished results.

45. This material has been further characterized by preliminary ¹¹³ CdNMR studies in the solid state (Kennedy, M. A.; Ellis, P. D.; Murai, T.;Sessler, J. L., unpublished results). The isotropic chemical shift ofthis complex (3·NO₃), σ=191 ppm relative to solid cadmium perchlorate,is shielded by ≅200-300 ppm relative to "normal" cadmium porphyrins suchas CdTPP²⁵ (σ=399 ppm⁴⁶) or CdPPIXDME²⁵ ((σ=480 ppm⁴⁷). This differencemay reflect the increased shielding caused by the presence of anadditional pair of electrons within the binding core of the "expanded""texaphyrin" ligand. A simulation of magic angle spinning spectra, usingthe theory of Maricq and Waugh,⁴⁸ yields an anisotropy of Δσ=207.6 andasymmetry, η=0.01, indicative of a system with a≧3-fold axis ofsymmetry. In addition, the eigenvalues of the chemical shift tensor werefound to be σ₁₁ =120.6 ppm, σ₂₂ =123 ppm, and σ₃₃ =329.6 ppm.

46. Jakobsen, H. J. J. Am. Chem. Soc. 1982, 104, 7442-7542.

47. Kennedy, M. A.; Ellis, P. D., submitted to J. Biol. Chem.

48. Maricq, M.; Waugh, J. S. J. Chem. Phys. 1979, 70, 3300-3316.

49. This data could also be analyzed using the iterative approach usedfor pyridine complexation. Values of K₁ and K₁ K₂ of 2.0×10⁴ M⁻¹ and1.9×10⁵ M⁻² were obtained using this approach.

50. SHELXTL-PLUS. Nicolet instrument Corporation, Madison, Wis., U.S.A.:1987.

51. SHELX76. A program for crystal structure determination. Sheldrick,G. M.; Univ. of Cambridge, England: 1976.

52. Cromer, D. T.; Mann, J. B. Acta Cryst. 1968, A24, 321-324.

53. Cromer, D. T.; Liberman, D. J. Chem. Phys. 1970, 53, 1891-1898.

54. Stewart, R. F., Davidson, E. R.; Simpson, W. T. J. Phys. Chem. 1965,42, 3175-3187.

55. International Tables for X-ray Crystallography, 1974, Vol. IV, p 55,Birmingham: Kynoch Press: 1974.

56. Cordes, A. W., personal communication (1983).

57. Gadol, S. M.; Davis, R. E. Organometallics 1982, 1, 1607-1613.

EXAMPLE 3

Gadolinium(III) complexes derived from strongly binding anionic ligands,such as diethylenetriamine pentaacetic acid (DTPA),¹,2,31,4,7,10-tetraazacyclododecane N,N',N",N'"-tetraacetic acid (DOTA),¹,4,5and 1,10-diaza-4,7,13,16-tetraoxacyclooctadecane-N,N'-diacetic acid(dacda),¹,6 are among the most promising of the paramagnetic contrastcurrently being developed for use in magnetic resonance imaging (MRI).¹Indeed, [Gd·DTPA]⁻ is now undergoing clinical trials in the UnitedStates for possible use in enhanced tumor detection protocols.¹Nonetheless, the synthesis of other gadolinium(III) complexes remains ofinterest since such systems might have greater kinetic stability,superior relaxivity, or better biodistribution properties than theexisting carboxylate-based contrast agents. One approach currently beingpursued is based on using water-soluble porphyrin derivatives, such astetrakis(4-sulfonatophenyl)porphyrin (TPPS).⁷,8,9 Unfortunately, thelarge gadolinium(III) cation cannot be accommodated completely¹⁰ withinthe relatively small prophyrin binding core (r=2.0 Å¹¹), and, as aconsequence, gadolinium porphyrin complexes are invariablyhydrolytically unstable.⁷,8,12,13 Larger porphyrin-like ligands,however, might offer a means of circumventing this problem.¹⁴⁻²²

As previously described, the present invention involves²³ the synthesisof a above "expanded porphyrin" system, 1_(B) (to which the trivial name"texaphyrin" has been assigned²⁴), and the structure of the bispyridineadduct of its cadmium(II) complex 2_(B). See FIG. 17A and FIG. 17B forthe structures of compound or complex 1_(B) -11_(B). The presence inthis structure of a near circular pentadentate binding core which isroughly 20% larger than that of the porphyrins,²³ coupled with therealization that almost identical ionic radii pertain for hexacoordinateCd²⁺ (r=0.9 Å) and Gd³⁺ (r=0.94 Å),²⁵ prompted exploration of thegeneral lanthanide binding properties of this new monoanionicporphyrin-like ligand. The synthesis and characterization of awater-stable gadolinium(III) complex (7_(B)) derived formally from a new16,17-dimethyl substituted analogue (6_(B))²⁶ of the original "expandedporphyrin" system, as well as the preparation and characterization ofthe corresponding europium(III) and samarium(III) complexes 8_(B) and9_(B) (See FIG. 17B).

Electronic spectra were recorded on a Beckman DU-7 spectrophotometer. IRspectra were recorded, as KBr pellets, from 4000 cm⁻¹ to 600 cm⁻¹ on aPerkin-Elmer 1320 spectrometer. Low resolution fast atom bombardmentmass spectrometry (FAB MS) was performed at Austin using a Finnigan-MATTSQ-70 instrument and either 3-nitrobenzyl alcohol or glycerol/oxalicacid as the matrix; high resolution FAB MS analyses (ERMS) wereperformed at the Midwest Center for Mass Spectrometry using CsI as astandard. Elementary analyses were performed by Galbraith Laboratories.

Materials. All solvents and reagents were of reagent grade quality,purchased commercially, and used without further purification. Sigmalipophilic SEPHADEX (LH-20-100) and Merck type 60 (230-400 mesh) silicagel were used for column chromatography.

Preparation of Nd complex 3_(B). The sp³ form of the ligand 10²⁷ (50 mg,0.1 mmol) was stirred with neodymium nitrate pentahydrate (63 mg, 0.15mmol) and proton sponge (64 mg, 0.3 mmol) in chloroform/methanol (150ml, v/v 1/2) for one day. The dark green reaction mixture was pouredonto ice/water/ammonium chloride and extracted with chloroform. Theorganic layer was washed with aqueous ammonium chloride and concentratedunder reduced pressure. The complex was chromatographed through sephadexusing neat chloroform, chloroform/methanol (10:1), methanol, and water.The dark green band collected from methanol was concentrated andrecrystallized from chloroform/methanol/n-hexane (ratio of chloroform tomethanol is 1 to 2) to yield 13 mg of 3 (18%). For 3: UV/VIS (CH₃ OH)λ_(max) (ε): 330.5 (33,096), 432.5 (85,762), 710.5 (10,724), 774.5(38,668); FAB MS (glycerol matrix): m/e (relative intensity) 631 (¹⁴²Nd, 95), 633 (¹⁴⁴ Nd, 100), 635 (¹⁴⁶ Nd, 77); IR (KBr) ν 3360, 2965,2930, 2870, 1610, 1560, 1450, 1400, 1350, 1250, 1205, 1135, 1080, 1050,980, 940, 905, 755 cm⁻¹.

The preparation of Sm complex 4_(B) was as follows. The macrocyle 10_(B)²⁷ (40 mg, 0.08 mmol) was stirred with platinum oxide (18 mg. 0.08 mmol)and samarium acetate hydrate (69 mg, 0.2 mmol) under reflux inbenzene/methanol (50 ml, v/v, 1/1). After two hours the reaction mixturewas filtered through celite and concentrated under reduced pressure. Theconcentrate was purified by chromatography through. Sephadex using onlychloroform as an eluent. After discarding a red band, a green band wascollected, concentrated in vacuo, and recrystallized fromchloroform/n-hexane to give 0.8 mg of 4 (ca. 1%). For 4_(B) : UV/VISλ_(max) nm 438, 706.5, 769; FAB MS (3-nitrobenzyl alcohol matrix): m/e(relative intensity) 635 (¹⁴⁷ Sm, 78), 636 (¹⁴⁹ Sm, 72), 637 (¹⁴⁹ Sm,73), 640 (¹⁵² Sm, 100), 642 (¹⁵⁴ Sm, 55).

The preparation of Eu complex 5_(B) was as follows. The macrocycle 10²⁷(50 mg, 0.1 mmol) was stirred with europium acetate hydrate (34 mg, 0.1mmol) and proton sponge (64 mg, 0.3 mmol) in chloroform/methanol (150ml, v/v, 1/2) for one day. The reaction mixture was poured ontoice/water and extracted with chloroform. The organic layer was washedwith aqueous ammonium chloride then concentrated and recrystallized fromchloroform/n-hexane. The recrystallized solid was purified by columnchromatography through sephadex using neat chloroform and neat methanolas eluents. The dark green band collected in methanol was concentratedto yield a small amount of a dark green solid (<1%). For 5: UV/VISλ_(max) nm 438, 700, 765; FAB MS (3-nitrobenzyl alcohol matrix): m/e(relative intensity) 639 (¹⁵¹ Eu, 94), 641 (¹⁵³ Eu, 100).4,5,9,24-Tetraethyl-10,16,17,23-tetramethyl-13,20,25,26,27-pentaazapentacyclo[20.2.1.1³,6.1⁸,11.0¹⁴,19]heptacosa-3,5,8,10,12,14(19),15,17,20,22,24-undecene (11_(B)). Thismacrocycle was prepared in ca. 90% yield from1,2-diamino-3,4-dimethylbenzene and2,5-bis-(3-ethyl-5-formyl-4-methylpyrrol-2-ylmethyl)-3,4-diethylpyrroleusing the acid catalyzed procedure reported earlier for the preparationof 10_(B).²⁷ For 11: mp 200° C. dec; ¹ H NMR β1.06 (6H, t, CH₂ CH₃),1.13 (6H, t, CH₂ CH₃) 2.15 (6H, s, phenyl-CH₃), 2.22 (6H, s,pyrrole-CH₃), 2.38 (4H, q, CH₂ CH₃), 2.50 (4H, q, CH₂ CH₃), 3.96 (4H, s,pyrrole₂ -CH₂), 7.19 (2H, s, aromatic), 8.10 (2H, s, CHN), 11.12 (1H, s,NH), 12.48 (2H, s, NH); ¹³ C NMR δ9.49, 15.33, 16.47, 17.22, 17.71,19.52, 22.41, 117.84, 120.40, 120.75, 125.11, 125.57, 134.95, 135.91,141.63; UV/VIS λ_(max) 367 nm; FAB MS, M⁺ 522; HRMS, M⁺ 521.35045 (calc.for C₃₄ H₄₃ N₅ 521.35185).

Preparation of Gd complex 7_(B). The sp³ form of ligand 11 (42 mg, 0.08mmol) was stirred with gadolinium acetate tetrahydrate (122 mg, 0.3mmol) and proton sponge (54 mg, 0.25 mmol) in chloroform/methanol (150ml, v/v 1/2) for one day. The dark green reaction mixture wasconcentrated under reduced pressure and chromatographed through silicagel (25 cm.×1.5 cm.) which was pretreated with chloroform/triethylamine(50 ml, v/v 25/1). Chloroform/triethylamine (25/1) andchloroform/methanol/triethylamine 25/2.5/1 v/v) was used as eluents. Adark red band was first collected followed by two green bands. The lastgreen band, which showed a clear aromatic pattern by UV/VIS, wasconcentrated and recrystallized from chloroform/n-hexane to give 14 mg(22%) of the Gd complex 7_(B). For 7_(B) : FAB MS (methanol/oxalicacid/glycerol matrix): m/e (relative intensity) 671 (¹⁵⁵ Gd, 58), 672(¹⁵⁶ Gd, 78), 673 (¹⁵⁷ Gd, 94), 674 (¹⁵⁸ Gd 100), 676 (¹⁶⁰ Gd, 64);HRMS, M⁺ 674.2366 (calc. for C₃₄ H₃₈ N₅ ¹⁵⁸ Gd 674 2368): UV/VIS (CHCl₃)λ_(max) nm (ε) 339.5 (14,850), 450.5 (36,350), 694.5 (6,757), 758.0(23,767); IR (KBr) ν 2990, 2960, 2900, 2830, 2765, 2700, 2620, 2515,1710, 1550, 1440, 1410, 1395, 1365, 1265, 1220, 1180, 1150, 1105, 1090,1060, 1040, 1095, 1045, 1015, 680 cm⁻¹ ; Anal. calc. for C₃₄ H₃₈ N₅Gd·(OH)₂ ·2H₂ O: C, 54.89; H, 5.96; N, 9.41. Found: C, 54.49; H, 5.95;N, 8.97.

The preparation of Eu complex 8_(B) was carried out. The macrocycle11_(B) (53 mg, 0.1 mmol) was stirred with europium acetate hydrate (105mg, 0.3 mmol) and proton sponge (64 mg, 0.3 mmol) in chloroform/methanol(150 ml, v/v 1/2) for 6 hrs. The dark green reaction mixture wasconcentrated under reduced pressure as described above with oneexception. Chloroform/triethylamine (25:1) andchloroform/methanol/triethylamine (25:5:1) were used as eluents. Thegreen complex 8 was recrystallized from chloroform/n-hexane to yield 26mg of product (33%). For 8_(B) : UV/VIS (CHCl₃) λ_(max) nm (ε) 339.5(24,570), 450.5 (63,913), 696.0 (10,527), 759.0 (40,907); FAB MS(methanol/oxalic acid/glycerol matrix): m/e (relative intensity) 667(¹⁵¹ Eu, 79), 669 (¹⁵³ Eu, 100); HRMS, M⁺, 669.2336 (calc for C₃₄ H₃₈ N₅¹⁵³ Eu 669.2340); IR (KBr) ν 2970, 2930, 2870, 2740, 2680, 2600, 2500,1700, 1535, 1430, 1350, 1255, 1205, 1165, 1135, 1095, 1075, 1050, 1030,980, 900 cm⁻¹ ; Anal. calc. for C₃₄ H₃₈ N₅ Eu·(OH)₂ O: C, 56.66; H,5.87; N, 9.72. Found: C, 55.92; H, 5.47; N, 9.95.

The preparation of the Sm³⁺ complex 9_(B) was as follows. The sp³ formof the ligand (11_(B)) (52 mg, 0.1 mmol) was stirred with samariumacetate hydrate (103.5 mg, 0.3 mmol) and proton sponge (64 mg, 0.3 mmol)in chloroform/methanol (150 ml, v/v 1/2) for one day. The dark greenreaction mixture was concentrated and purified by silica gelchromatography as described above. The resulting crude material was thenrecrystallized from chloroform/n-hexane to give 29 mg of 9 in 37% yield.For 9: UV/VIS (CHCl₃) λ_(max) nm (ε) 339.5 (21,617), 451.0 (56,350),695.5 (9,393), 760.0 (35,360); FAB MS (3-nitrobenzyl alcohol): m/e(relative intensity) 663 (¹⁴⁷ Sm, 74.8), 664 (¹⁴⁸ Sm, 82.3), 665 (¹⁴⁹Sm, 84.58), 668 (¹⁵² Sm, 100, 670 (¹⁵⁴ Sm, 78.5); HRMS, M⁺, 668.2300(calc. for C₃₄ H₃₈ N₅ ¹⁵² Sm 668.2322); IR (KBr) ν 2990, 2950, 2890,2760, 2700, 2620, 2520, 1720, 1620, 1550, 1440, 1360, 1265, 1215, 1175,1145, 1105, 1085, 1060, 995, 945, 910, 680 cm⁻¹ ; Anal. calc for C₃₄ H₃₈N₅ Sm·(OH)₂ ·O: C, 54.08; H, 6.14; N, 9.27. Found: C, 54.30; H, 5.66; N,9.06.

As described earlier,²³ (see Example 1) treatment of themethylene-bridged, or sp³ form of the texaphyrin macrocycle 10_(B) withCd(II) salts in air saturated methanol/chloroform at ambient temperatureleads to the formation of the green Cd(II) complex 2 in roughly 25%yield, with both metal insertion and oxidation taking place concurrentlyunder the reaction conditions. When a similar procedure was carried outusing a variety of trivalent lanthanide salts [i.e. Ce(OTf)₃, Pr(OAc)₃,Nd(NO₃)₃, Sm(OAc)₃, Eu(OAc)₃, Gd(OAc)₃, DY(OTf)₃, TbCl₃, Er(OTf)₃,Tm(NO₃)₃, and Yb(NO₃)₃ ] no metal complexes of 1 (or 10) were obtained(as judged by the absence of changes in the UV/visible spectrum). If,however, N,N',N",N'"-tetramethyl-1,8-diaminonaphthalene ("protonsponge") was added to the various reaction mixtures, the high energy,low intensity band of 10 at λ_(max) =365 nm disappeared over the courseof several hours to several days (depending on the salt in question) andwas replaced by two strong transitions in the 435-455 nm (Soret) and760-800 nm (Q-band) regions, suggesting that ligand oxidation and metalbinding had occurred.²⁸ Unfortunately, isolation of these putativemetal-containing products proved problematic: Direct chromatography oneither silica gel or lipophilic Sephadex in general gave only smallquantities of metal-free oxidized ligand 1_(B) and essentially none ofthe desired metalated material. Indeed, only in the case of thesamarium(III) acetate salt did it prove possible to isolate a tracequantity (ca. 1% yield) of the desired complexes (4) by chromatographyon Sephadex. It was interesting to find, however, that a dark greenneodymium(III) complex 3_(B) could be obtained in almost 20% yield byquenching the reaction mixture with ice water, extracting repeatedlywith chloroform, washing with aqueous ammonium chloride, purifying bychromatography on Sephadex, and recrystallizing fromchloroform/methanol/n-hexane. Unfortunately this work-up procedureproved ineffective in the case of the other putative lanthanidecomplexes (including, unfortunately, that derived from Gd³⁺), althoughit did prove possible to obtain trace quantities of the europium(III)complex (5_(B)) using this procedure.

Since spectral evidence suggested that metal uptake and ligand oxidationwere occurring when the sp³ macrocycle 10_(B) was treated with numerousother Ln³⁺ salts, it was puzzling that only the neodymium(III) complex(3) could be isolated in reasonable yield. A careful analysis suggestedthat, in certain instances, notably Sm³⁺, Eu³⁺, Gd³⁺, the problem wasnot due to hydrolytic instability Rather, it derived from the very highwater solubility of the lanthanide complexes which precludedreextraction back into organic solvents following the initial aqueouswashes! This observational hypothesis led to the consideration that morehydrophobic texaphyrin analogues would prove valuable in the preparationand isolation of "expanded porphyrin" in lanthanide complexes.

To test the above assumption, a simple dimethylated analogue (11_(B)) ofthe original sp³ hybridized ligand 10_(B) was prepared. This new, morehydrophobic, sp³ hybridized ligand was obtained in ca. 90% yield bycondensing 1,2-diamino-4,5-dimethylbenzene with2,5-Bis-(3-ethyl-5-formyl-4-methylpyrrol-2-ylmethyl)-3,4-diethylpyrroleunder acid catalyzed conditions identical to those used to prepare 10.²⁷Treatment of this texaphyrin precursor with Gd(OAc)₃, Eu(OAc)₃, andSm(OAc)₃ under reaction and work-up conditions similar to those used toobtain 3_(B), then gave the cationic complexes 7_(B), 8_(B), and 9_(B),as their dihydroxide adducts,²⁹ in 22%, 33%, and 37% yieldsrespectively! It appears that these increased yields derive directlyfrom the increased hydrophobicity of the new dimethyl substitutedtexaphyrin ligand system (6_(B)).

The new lanthanide complexes reported here are unique in several ways.For instance, as judged by fast atom bombardment mass spectrometric (FABMS) analysis, complexes 3_(B) -5_(B) and 7_(B) -9_(B) are mononuclear1:1 species, a conclusion that is further supported, in the case ofcompounds 7_(B) -9_(B), by both high resolution FAB MS accuratemolecular weight determinations and combustion analysis. In other words,we have found no evidence of 1:2 metal to ligand "sandwich" systems, orhigher order combinations as are often found in the case of the betterstudied lanthanide porphyrins.³¹

The electronic spectra represents a second remarkable feature of thesenew materials: All six lanthanide complexes isolated to date display adominant Soret-like transition in the 435-455 nm region which isconsiderably less intense than that observed in the correspondingmetalloporphyrins (c.f. FIG. 18A and FIG. 18B),⁷ and show a prominentlow energy Q-type band in the 760-800 nm region. This latter feature isdiagnostic of this class of 22 π-electron "expanded porphyrins"²³ and isboth considerably more intense and substantially red-shifted (by ca. 200nm!) as compared to the corresponding transitions in suitable referencelanthanide prophyrins (e.g., [Gd.TPPS]⁺, λ_(max) ≈575 nm⁷). Within thecontext of these general observations, it is interesting to note thatcomplexes derived from the somewhat more electron rich ligand 6_(B) alldisplay Q-type bands that are blue shifted by ca. 5-15 nm as compared tothose obtained from the original texaphyrin 1_(B).

A third notable property of complexes 7_(B) -9_(B) is their highsolubility in both chloroform and methanol. The fact that these threecomplexes are also moderately soluble (to roughly 10⁻³ M concentrations)in 1:1 (v.v.) methanol/water mixtures was of particular interest.Moreover, as initially suggested on the basis of the preliminary studieswith 3-5 discussed above, these materials are stable to these solventconditions. For instance, a 3.5×10⁻⁵ M solution of the gadoliniumcomplex 7_(B) in 1:1 (v.v.) methanol/water at ambient temperature showsless than 10% bleaching of the Soret and Q-type bands when monitoredspectroscopically over the course of 2 weeks. This suggests that thehalf-life for decomplexation and/or decomposition of this complex is≧100 days under these conditions. Under the conditions of the experimentdescribed above, no detectable shifts in the position of the Q-type bandare observed yet the Q-type transition of the free-base 6_(B) falls ca.20 nm to the blue of that of 7_(B),³⁰ while shifts in this directionwould be expected if simple demetalation were the dominant pathwayleading to the small quantity of observed spectral bleaching.

The strong hydrolyric stability of complexes 7_(B) -9_(B) is in markedcontrast to that observed for simple, water soluble gadoliniumporphyrins, such as [Gd·TPPS]⁺, which undergo water-induced demetalationin the course of several days when exposed to an aqueous environment.⁷,8It thus appears likely that gadolinium(III) complexes derived from thenew texaphyrin ligand 6_(B), or its analogues, should provide the basisfor developing new paramagnetic contrast reagents for use in MRIapplications. In addition, the ease of preparation and stablemononuclear nature of complexes 7_(B) -9_(B) suggests that such expandedporphyrin ligands might provide the basis for extending further therelatively underdeveloped coordination chemistry of the lanthanides.

Literature citations in the following list are incorporated by referenceherein for the reasons cited.

REFERENCES

1. For a recent review see: Lauffer, R. B. Chem. Rev. 1987, 87, 901-927.

2. Kornguth, S. E.; Turski, P. A.; Perman, W. H.; Schultz, R.; Kalinke,T.; Reale, R.; Raybaud, F. J. Neurosurg. 1987, 66, 898-906.

3. Koenig, S. H.; Spiller, M.; Brown, R. D.; Wolf, G. L. Invest. Radiol.1986, 21, 697-704.

Cacheris, W. P.; Nickle, S. K.; Sherry, A. D. Inorg. Chem. 1987, 26,958-960.

5. (a) Loncin, M. F.; Desreux, J. F.; Merciny, E. Inorg. Chem. 1986, 25,2646-2648. (b) Spirlet, M.-R.; Rebizant, J.; Desreux, J. F.; Loncin,M.-F. Inorg. Chem. 1984, 23, 359-363.

6. (a) Chang, C. A.; Sekhar, V. C. Inorg. Chem. 1987, 26, 1981-1985. (b)Chang, C. A.; Ochaya, V. O. Inorg. Chem. 1986, 25, 355-358. (c) Chang,C. A.; Rowland, M. E. Inorg. Chem. 1983, 22, 3866-3869.

7. Horrocks, W. D.; Hove, E. G. J. Am. Chem. Soc. 1978, 100, 4386-4392.

8. Lyon, R. C.; Faustino, P. J.; Cohen, J. S.; Katz, A.; Mornex, F.;Colcher, D.; Baglin, C.; Koenig, S. H.; Hambright, P. Magn. Reson. Med.1987, 4, 24-33.

9. Radzki, S.; Krauz, P.; Gaspard, S.; Giannotti, C. Inorg. Chim. Acta1987, 138, 139-143.

10. Buchler, J. W. in "The Porphyrins," Dolphin D. ed., Academic Press,New York, 1978, Vol. 1, Chapter 10.

11. Hoard, J. L. in "Porphyrins and Metalloporphyrins"; Smith, K., Ed;Elsevier, Amsterdam, 1975, Chapter 8.

12. (a) (Horrocks, W. D., Jr.; Wong, C.-P. J. Am. Chem. Soc. 1976, 98,7157-7162. (b) Wong, C.-P.; Venteicher, R. F.; Horrocks, W. D., Jr. J.Am. Chem. Soc. 1974, 96, 7149-7150.

13. Srivastava, T. S. Bioinorg. Chem. 1978, 8, 61-76.

14. Although several large porphyrin-like aromatic macrocycles,including the "sapphyrins",¹⁵,16, "platyrins",¹⁷ "pentaphyrin",¹⁸ and"[26]porphyrin"¹⁹ have been prepared in their metal-free forms, and auranyl complex has been stabilized with a large "superphthalocyanine",²⁰we are not aware of any lanthanide complexes formed from thesesystems.²¹

15. Bauer, V. J.; Clive, D. R.; Dolphin, D.; Paine, J. B. III; Harris,F. L.; King, M. M.; Loder, J.; Wang, S.-W. C.; Woodward, R. B. J. Am.Chem. Soc. 1983, 105, 6429-6436.

16. Broadhurst, M. J.; Grigg, R.; Johnson, A. W. J. Chem. Soc. PerkinTrans. 1, 1972, 2111-2116.

17. (a) Berger, R. A.; LeGoff, E. Tetrahedron Lett. 1978, 4225-4228. (b)LeGoff, E.; Weaver, O. G. J. Org. Chem. 1987, 710-711.

18. (a) Rexhausen, H.; Gossauer, A. J. Chem. Soc., Chem. Commun. 1983,275. (b) Gossauer, A. Bull. Soc. Chim. Belg. 1983, 92, 793-795.

19. Gosmann, M.; Franck, B. Angew. Chem. 1986, 98, 1107-1108; Angew.Chem. Int. Ed. Eng. 1986, 25, 1100-1101.

20. (a) Day, V. W.; Marks, T. J.; Wachter, W. A. J. Am. Chem. Soc. 1975,97, 4519-4527. (b) Marks, T. J.; Stojakovic, D. R. J. Am. Chem. Soc.1978, 100, 1695-1705. (c) Cuellar, E. A.; Marks, T. J. Inorg. Chem.1981, 20, 3766-3770.

21. Sessler, J. L; Cyr, M.; Murai, T. Comm. Inorg. Chem., in press.

22. For examples of lanthanide cationic complexes stabilized by moreconventional Schiff base macrocycles see for instance: (a) Backer-Dirks,J. D. J.; Gray, C. J.; Hart, F. A.; Hursthouse, M. B.; Schoop, B. C. J.Chem. Soc., Chem. Commmun. 1979, 774-775. (b) De Cola, L.; Smailes, D.L.; Vallarino, L. M. Inorg. Chem. 1986, 25, 1729-1732. (c) Sabbatini,N.; De Cola, L.; Vallarino, L. M.; Blasse, G. J. Phys. Chem. 1987, 91,4681-4685. (d) Abid, K. K.; Fenton, D. E.; Casellato, U.; Vigato, P.;Graziani, R. J. Chem. Soc., Dalton Trans. 1984, 351. (e) Abid, K. K.;Fenton, D. E. Inorg. Chim. Acta 1984, 95, 119-125. (f) Sakamoto, M. BullChem. Soc. Jpn. 1987, 60, 1546-1548.

23, Sessler, J. L.; Murai, T.; Lynch, V.; Cyr, M. J. Am. Chem. Soc.1988, 110, 5586-5588.

24. Chemical & Engineering News Aug. 8, 1988, 26-27.

25. Cotton, F. A.; Wilkinson, G. "Advanced Inorganic Chemistry, 4thed.," John Wiley, New York, 1980, pp. 589 and 982.

26. The systematic name for this compound is4,5,9,24-tetraethyl-10,16,17,23-tetramethyl-13,20,25,26,27-pentaazapentacyclo-[20.2.1.1³,6.1⁸,11.0¹⁴,19]heptacosa-1,3,5,7,9,11(27),12,14,16,18,20,22(25),23-tridecaene.

27. Sessler, J. L.; Johnson, M. R.; Lynch, V. J. Org. Chem. 1987, 52,4394-4397.

28. The relation between the optical bands (nm) observed just after thereaction and the trivalent lanthanide cation employed are as follows.Ce: 453, 782; Pr: 437, 797; Nd: 439, 786; Sm: 438, 769; Eu: 438, 765;Gd: 438, 765; Tb: 439, 764; Dy: 438, 765; Tm: 437, 765; Yb: 437, 764.

29. As judged by the IR and microanalytical data, under the reaction andwork up conditions, hydroxide anions serve to displace the acetateligands presumably present following the initial metal insertionprocedure. Similar exchanges have also observed in the case of thecadmium complex 2 (prepared from Cd(OAc)₂) where ¹ H NMR analyses can bemade with ease.³⁰

30. Murai, T.; Hemmi, G.; Sessler, J. L., unpublished results.

31. (a) Buchler, J. W.; Cian, A. D.; Fischer, J.; Kihn-Botulinski, M.;Paulus, H.; Weiss, R. J. Am. Chem. Soc. 1986, 108, 3652-3659. (b)Buchler, J. W.; Cian, A. D.; Fischer, J.; Kihn-Botulinski, M.; Weiss, R.Inorg. Chem. 1988, 27, 339-345. (c) Buchler, J. W.; Scharbert, B. J. Am.Chem. Soc. 1988, 110, 4272-4276. (d) Buchler, J. W.; Kapellmann, H.-G.;Knoff, M.; Lay, K.-L.; Pfeifer, S. Z. Naturforsch. 1983, 38b, 1339-1345.

EXAMPLE 4

The photophysical properties of a new series oftripyrroledimethine-derived "expanded porphyrins" ("texaphyrins") arereported; these compounds show strong low energy optical absorptions inthe 730-770 nm spectral range as well as a high triplet quantum yield,and act as efficient photosensitizers for the production of singletoxygen in methanol solution.

Photodynamic therapy is among the more promising of modalities currentlybeing considered for the treatment of localized neoplasia¹ anderadication of viral contaminants in blood.² As a result, considerableeffort has been devoted to the development of effectivephotochemotherapeutic agents.³ To date, porphyrins and theirderivatives, phthalocyanines, and naphthalocyanines have been among themost widely studied compounds in this regard. Unfortunately, all ofthese dyes suffer from critical disadvantages. While porphyrinderivatives have high triplet yields and long triplet lifetimes (andconsequently transfer excitation energy efficiently to tripletoxygen),^(3b),3g their absorption in the Q-band region often parallelsthat of heme-containing tissues. Phthalocyanines and naphthalocyaninesabsorb in a more convenient spectral range but have significantly lowertriplet yields;⁴ moreover, they tend to be quite insoluble in polarprotic solvents, and are difficult to funtionalize. Thus at present thedevelopment of more effective photochemotherapeutic agents appears torequire the synthesis of compounds which absorb in the spectral regionwhere living tissues are relatively transparent (i.e., 700-1000nm),^(1d) have high triplet quantum yields, and are minimally toxic. Thepresent inventors have recently reported⁵ (see Example 1) the synthesisof a new class of aromatic prophyrin-like macrocycles, thetripyrroledimethine-derived "texephyrins", which absorb strongly in thetissue-transparent 730-770 nm range. The photophysical properties ofmetallotexaphyrins 1_(C) -7_(C) parallel those of the correspondingmetallaporphyrins and the diamagnetic complexes 1_(C) -4_(C) sensitizethe production of ¹ O₂ in high quantum yield. FIG. 19 shows theschematic structure, metal complexes and derivatives of compounds of thepresent invention (1_(C) -7_(C)).

The absorption spectrum of 1_(C) ·Cl is shown in FIG. 20A. Thisspectrum, which is representative of this class of compounds (cf. Table3), is characterized by strong Soret- and Q-type bands, the latter beingof particular interest The fluorescence excitation spectrum of thiscomplex, monitored at the emission maximum (ca. 780 nm; see FIG. 20B),and the absorption spectrum are superimposable in the visible region(370-800 nm) showing that internal conversion to the first excitedsinglet state is quantitative upon photoexcitation in the Soret orQ-band regions. While the fluorescence quantum yields (φ_(f)) for 1_(C)-4_(C) are only 0-1%, the quantum yields for triplet formation (φ_(t))of these diamagnetic metalltexaphrins can approach unity and resemblethose found for metalloporphyrins.⁶ The triplet-triplet transientspectrum of 1_(C) ·Cl , given in FIG. 21B, shows bleaching in the Soret-and Q-bands of the ground state and a positive absorbance change in the450-600 nm region, again reminiscent of metalloporphyrin tripletspectra.⁷ FIG. 21A shows the decay of this triplet state in deoxygenatedmethanol, from which a lifetime (τ_(t)) of 67 μs is calculated. Similartriplet spectra, lifetimes, and quantum yields were found for otherdiamagnetic metallotexaphyrin derivatives in methanol and for 1_(C) ·Clin mixed methanol-water solutions. Interestingly, low temperaturephosphorescence could not be observed for any of the compounds inmethanol glasses. Finally, several complexes containing paramagneticmetal ions (e.g. Mn^(II), Sm^(III), and Eu^(III), structures 5_(C)-7_(C)) were investigated. They proved to be non-luminescent and theirtriplet excited states could not be detected with our laser flashphotolysis set-up, which has a time resolution of ca. 10 ns.

In methanol solution, the triplet excited states of 1_(C) -4_(C) werequenched by molecular oxygen with biomolecular rate constants of(2.6±0.2)×10⁹ dm³ mol⁻¹ s⁻¹. In aerated solution, the triplet statedecay profile could be described in terms of a single exponentialprocess with an average lifetime of (175±20) ns; thus, interactionbetween the triplet species and O₂ is quantitative. Laser excitation(355 nm, 80 mJ, 10 ns) of the compound in aerated methanol gave no redoxproducts (e.g. texaphyrin cation and superoxide anion) but, using a Gediode,⁸ the production of ¹ O₂ was observed clearly from itscharacteristic luminescence at 1270 nm. This luminescence decayed with alifetime of 12.5±0.3 μs and its initial intensity, as extrapolated tothe centre of the laser pulse, was a linear function of the number ofphotons absorbed by the texaphyrin complex. Comparison of the initialintensity with that obtained using tetrakis (4-hydroxyphenyl)porphyrin(THPP) as photosensitizer^(3b) under identical conditions allowedcalculation of the quantum yields for production of singlet oxygen(.sup.Φ.sbsp.1_(o).sbsb.2). The derived values are seen to parallel thetriplet quantum yields (Table 3); the triplet state reaction appears topartition between generation of ¹ O₂ (74-78%) and formation ofvibrationally excited O₂ (22-26%). These .sup.Φ.sbsp.1_(o).sbsb.2 valuescompare favourably with those observed with porphyrins^(3b) and are muchsuperior to values obtained with phthalocyanines and naphthalocyanines⁴due to the improved triplet state yields. Thus diamagnetic texaphyrincomplexes appear to be highly efficient photosensitizers for theformation of ¹ O₂.

                                      TABLE 3                                     __________________________________________________________________________    Optical and photophysical properties of metallotexaphyrins in CH.sub.3        OH.                                                                                 Absorption                                                                            Emission                                                                             Φ.sub.f                                                                         Φ.sub.t                                                                        τ.sub.t                                                                       Φ.sup.1.sub.O2                        Complex                                                                             λ.sub.max (nm                                                                  λ.sub.max (nm)                                                                (±10%)                                                                           (±5%)                                                                           (μs)                                                                           (±10%)                                                                           S.sup.(a)                           __________________________________________________________________________    1.sub.C Cl                                                                          410 732 767    0.013 0.82 67  0.61  0.74                                2.sub.C Cl                                                                          412 738 765    0.012 0.88 37  0.65  0.73                                3.sub.C NO.sub.3                                                                    417 759 780    0.011 0.88 55  0.69  0.78                                4.sub.C NO.sub.3                                                                    421 760 788    0.009 0.97 36  0.74  0.76                                5.sub.C OH                                                                          420 760 --     <0.001                                                                              ND.sup.(b)                                                                         --  <0.05 --                                  6.sub.C (OH).sub.2                                                                  450 763 --     <0.001                                                                              ND   --  <0.05 --                                  7.sub.C (OH).sub.2                                                                  451 762 --     <0.001                                                                              ND   --  <0.05 --                                  SiNC.sup.(c)                                                                        310 776 780    --    0.39 331 0.35  0.90                                __________________________________________________________________________     .sup.(a) S = .sup.Φ.sbsp.1 O.sub.2 /Φ.sub.t.                          .sup. (b) ND = not detected                                                   .sup.(c) SiNC = bis(trin-hexylsiloxy)(2,3-naphthalocyaninato) silicon in      benzene; see reference 4.                                                

In summary, the new metallotexaphrin complexes discussed here show threeimportant optical properties which make them unique among existingporphyrin-like macrocycles. They absorb strongly in a physiologicallyimportant region (i.e. 730-770 nm), form long-lived triplet states inhigh yield, and act as efficient photosensitizers for the formation ofsinglet oxygen (see, e.g. FIG. 21A and FIG. 21B). These properties,coupled with their high chemical stability and appreciable solubility inpolar media, suggest that these cationic complexes could serve as viablephotosensitizers in emerging photodynamic protocols. Preliminary invitro studies of 3_(C) ·NO₃ in 10% human serum, in which a significantdecrease in herpes simplex (HSV-1) infectivity and lymphocyte mitogenicactivity are observed upon irradiation at 767 nm,⁹ affirm thefeasibility of this approach.

REFERENCES

1. (a) For an overview see: C. J. Gomer, Photochem. Photobiol. 1987, 46,561 (this special issue is entirely devoted to this topic). See also:(b) T. J. Dougherty, Photochem. Photobiol. 1987, 45, 879; (c) A. R.Oseroff, D. Ohuoha, G. Ara, D. McAuliffe, J. Foley, and L. Cincotta,Proc. Natl. Acad. Sci. U.S.A., 1986, 83, 9729; (d) S. Wan, J. A.Parrish, R. R. Anderson, and M. Madden, Photochem. Photobiol., 1981, 34,679; (e) A. Dahlman, A. G. Wile, R. B. Burns, G. R. Mason, F. M.Johnson, and M. W. Berns, Cancer Res., 1983, 43, 430.

2. J. L. Matthews, J. T. Newsam, F. Sogandares-Bernal, M. M. Judy, H.Skiles, J. E. Levenson, A. J. Marengo-Rowe, and T. C. Chanh,Transfusion, 1988, 28, 81.

3. (a) M. R. Detty, P. B. Merkel, and S. K. Powers, J. Am. Chem. Sec.,1988, 110, 5920; (b) R. Bonnett, D. J. McGarvey, A. Harriman, E. J.Land, T. G. Truscott, and U.-J. Winfield, Photochem. Photobiol., 1988,48, 271; (c) R. Bonnett, S. Ioannou, R. D. White, U.-J. Winfield, and M.C. Berenbaum, Photobiochem. Photobiophys. 1987, Suppl., 45; (d) P. A.Scourides, R. M. Bohmer, A. H. Kaye, and G. Morstyn, Cancer Res., 1987,47, 3439; (e) M. C. Berenbaum, S. L. Akande, R. Bonnett, H. Kaur, S.Ioannou, R. D. White, and U.-J. Winfield, Br. J. Cancer, 1986, 54, 717;(f) J. D. Spikes, Photochem. Photobiol., 1986, 43, 691; (g) D. Kesseland C. J. Dutton, Photochem. Photobiol., 1984, 40, 403.

4. P. A. Firey and M. A. J. Rodgers, Photochem. Photobiol., 1987, 45,535.

5. (a) J. L. Sessler, T. Murai, V. Lynch, and M. Cyr, J. Am. Chem. Soc.,1988, 110, 5586. (b) J. L. Sessler, T. Murai, and G. Hemmi, submitted toInorg. Chem.

6. "The Porphyrins"; D. Dolphin, Ed., Academic Press: New York,1978-1979, Vols. I-VII.

7. A. Harriman, J. Chem. Soc., Faraday Trans. 2, 1981, 77, 1281.

8. M. A. J. Rodgers and P. T. Snowden, J. Am. Chem. Soc., 1982, 104,5541.

9. M. H. Judy, J. L. Matthews, G. Hemmi, J. L. Sessler, to be published.

EXAMPLE 5

Acquired immunodeficiency syndrome (AIDS) and cancer are among the mostserious public health problems facing our nation today. AIDS, firstreported in 1981 as occurring among male homosexuals,¹ is a fatal humandisease which has now reached pandemic proportions. Cancer, in spite ofsome very significant advances in diagnostics and treatment in recentyears, remains the third leading-cause of death in this country. Findingbetter ways to detect, treat, and reduce the transmission of thesedisorders are thus research objectives of the highest importance.

One of the more promising new modalities currently being explored foruse in the control and treatment of tumors is photodynamic therapy(PDT).¹⁻⁵ This technique is based on the use of a photosensitizing dye,which localizes at, or near, the tumor site, and when irradiated in thepresence of oxygen serves to produce cytotoxic materials, such assinglet oxygen (O₂ (¹ Δ_(g))), from otherwise benign precursors (e.g.(O₂ (³ Σ_(g) -)). Much of the current excitement associated with PDTderives from just this property: In marked contrast to current methods(e.g. conventional chemotherapy), in PDT the drugs themselves can (andshould) be completely innocuous until "activated" with light by anattending physician. Thus a level of control and selectivity may beattained which is not otherwise possible.

At present, diamagnetic porphyrins and their derivatives are consideredthe dyes of choice for PDT. It has been known for decades thatporphyrins, such as hematoporphyrin, localize selectively in rapidlygrowing tissues including sarcomas and carcinomas,⁶ although the reasonsfor this selectivity remain recondite. Currently most attention isfocusing on the so-called hematoporphyrin derivative (HPD),²⁻⁵,7-21 anincompletely characterized mixture of monomeric and oligomericporphyrins produced by treating hematoporphyrin dihydrochloride withacetic acid-sulfuric acid followed by dilute base.²²⁻²⁷ Fractions richin the oligomeric species, which are believed to have the besttumor-localizing ability,²³,26 are marketed under the trade namePhotofirin II® (PII) and are currently undergoing phase III clinicaltrials for obstructed endobronchial tumors and superficial bladdertumors. Here, the mechanism of action is thought to be largely, if notentirely, due to the photoproduction of singlet oxygen (O₂ (¹ Δ_(g))),although alternative mechanisms of action, including those involvingsuperoxide anion or hydroxyl and/or porphyrin-based radicals cannot beentirely ruled out.²⁸⁻³³

Singlet oxygen is also believed to be the critical toxic speciesoperative in experimental photsensitized blood purificationprocedures.³⁴⁻³⁹ This very new application of photodynamic therapy is oftremendous potential importance: It may provide a safe and effectivemeans of removing enveloped viruses such as HIV-1, herpes simplex (HSV),cytomegalovirus (CMV), various forms of hepatitis, as well as otheropportunistic blood-borne infections (e.g. bacteria and malariaplasmodium) from transfused whole blood. Given that AIDS is currently anineffectively treated and usually fatal disease, the benefit of such ablood purification procedure would be of inestimable value.

At present, sexual relations and needle-sharing are the dominantmechanisms for the spread of AIDS.¹ An increasing percentage of AIDSinfections, however, are now occurring as a result of bloodtransfusions.¹,40-43 Unfortunately, banked blood components areessential products for the practice of modern medicine and as a resultthis method of transmission is not likely to be precluded by simplechanges in lifestyle. Rather, an absolutely fail-proof means must bedeveloped to insure that all stored blood samples are free of the AIDSvirus (and ideally all other blood-borne pathogens). To a certain extentthis can be accomplished by screening the donors' histories and carryingout serologic tests. At present, however, the serologic tests for HIV-1are insufficient to detect all infected blood samples, in particular,those derived from donors who have contacted the disease but not yetproduced detectable antibodies.⁴²,43 In addition, new mutants of theAIDS virus have been detected; some or all of these may escape detectionby current means.¹ Thus, an antiviral system is needed which removes anyform of HIV-1 from stored blood. This is particularly important since astored blood sample from one infected donor could potentially end upbeing administered to several different patients, in, for instance, thecourse of pediatric care.

Ideally, any blood purification procedure used to remove AIDS virus orother blood-borne pathogens should operate without introducingundesirable toxins, damaging normal blood components, or inducing theformation of harmful metabolites. In general, this precludes the use ofcommon antiviral systems such as those based on heating, UV irradiation,or purely chemical means. A promising approach is the photodynamic onealluded to above. Here, preliminary studies, carried out bycollaborators at the Baylot Research Foundation, Dr. Matthews and histeam,³⁴⁻³⁷ and others,³⁸,39 have served to show that HPD and PII, in farlower dosages than are required for tumor treatment, can act asefficient photosensitizers for the photo-deactivation of cell-freeHIV-1, HIV, hepatitis and other enveloped viruses. On the basis ofavailable data, it is considered likely that the success of thisprocedure derives from the fact that these dyes localize selectively ator near the morphologically characteristic, and physiologicallyessential, viral membrane ("envelope") and catalyze the formation ofsinglet oxygen upon photoirradiation. The singlet oxygen so produced isbelieved, in turn, to destroy the essential membrane envelope. Thiskills the virus and eliminates infectivity. Photodynamic bloodpurification procedures, therefore, apparently rely on the use ofphotosensitizers which localize selectively at vital membranes, just asmore classic tumor treatments require dyes that are absorbed or retainedpreferentially at tumor sites. To the extent that this is true, simpleenveloped DNA viruses like HSV-1 will prove to be good models fortesting putative photosensitizers for potential use in killing the farmore hazardous HIV-1 retrovirus. It is important to note, however, thatthis correspondence holds only as far as freely circulating (as opposedto intracellular) viruses are concerned. Complete prophylactic removalof HIV-1 from blood products will require the destructive removal of thevirus from within monocytes and T lymphocytes..sup. 44

Critical as are the potential anti-tumor and anti-viral photodynamicapplications which are currently being explored using HPD and PII, it isimportant to realize that these photosensitizers are not ideal. Indeed,this "first generation" of dyes suffers from a number of seriousdeficiencies which may in fact militate against their eventual use inbiomedical applications. They contain a range of chemical species, theyare neither catabolized nor excreted rapidly from the body, and theyabsorb but poorly in the red part of the spectrum where blood and otherbodily tissues are most transparent.⁵ Each of these deficiencies can anddoes have important clinical consequences. For instance, the fact thatHPD and PII do not contain a single chemically well-defined constituent,coupled with the fact that the active components have yet to beidentified with certainty,²³⁻²⁷ means that the effective concentrationscan and often do vary from preparation to preparation. Thus the dosage,and the light fluence, cannot necessarily be optimized and predeterminedfor any particular application. Moreover, the fact that they are notmetabolized rapidly means that significant quantities of these dyes willremain in stored blood units after prophylactic photoinduced HIV-1removal and remain in patients' bodies long after photodynamic tumortreatment. The latter retention problem, in particular, is known to bequite serious: HPD and PII localize in the skin and inducephotosensitivity in patients for weeks after administration.⁵,45

It is, however, the last of the above shortcomings (the lack of a trulylow-energy transition) that is considered to be most serious: Becausethe longest wavelength absorption maximum for these dyes falls at 630nm, most of the incipient energy used in photo-treatment is dispersed orattenuated before reaching the center of a deep-seated tumor and as aresult little of the initial light is available for singlet oxygenproduction and therapy.⁴⁶⁻⁴⁸ Indeed, one study, while used a mouse modeland a 3 mm tumor implanted beneath the skin served to indicate that asmuch as 90% of the energy is lost by the base of the tumor.⁴⁶ Asillustrated by the data in FIG. 22, taken from ref. 47, far moreeffective treatment deep-seated or large tumors might be possible ifphotosensitizers could be developed which absorb in the >700 nm region,provided, of course, they retain the desirable features of HPD and PII(e.g. selective localization in target tissues and low dark toxicity).The present aspect of the invention involves development of suchimproved photosensitizers for use in photdynamic tumor treatment andblood purification protocols.

1. Easily available

2. Low intrinsic toxicity

3. Long wavelength absorption

4. Efficient photosensitizer for singlet oxygen production

5. Fair solubility in water

6. Selective-up-take in tumor tissue and/or

7. Showing high affinity for enveloped viruses

8. Quick degradation and/or elimination after use

9. Chemically pure and stable

10. Easily subject to synthetic modification

The list summarizes those features which would be desirable inbiomedical photosensitizers. Clearly, there is going to be somevariability in the requirements, depending on application. For instance,photosensitizers designed for use in blood purification protocols shouldbe designed to be less chemically stable than those used forphotodynamic therapy. The idea being, that following irradiation thedyes will undergo rapid degradation or hydrolysis to yield nontoxic andnonactive metabolites. For tumor treatment, greater stability appearsdesirable as longer times are apparently required to achieve selectivelocalization in the neoplastic tissues. In both cases, of course, lowtoxicity and good long-wavelength absorption and photosensitizationproperties are an absolute must.

In recent years, considerable effort has been devoted to the synthesisand study of new potential photosensitizers which might meet thesedesiderata. Although a few of these have consisted of classic dyes suchas those of the rhodamine and cyanine classes,⁴⁹⁻⁵¹ many have beenporphyrin derivatives with extended π networks.⁵⁶⁻⁶⁷ Included in thislatter category (See FIG. 23A, FIG. 23B, FIG. 23C and FIG. 23D) are thepurpurins⁵⁵ (e.g. 1_(D)) and verdins⁵⁶ (e.g. 2_(B)) of Morgan and otherchlorophyll-like species,⁵⁷⁻⁵⁹ the benz-fused prophyrins (3_(D)) ofDolphin et al.,⁶⁰ and the sulfonated phthalocyanines andnapthophthalocyanines (4_(D)) studied by Ben-Hur,⁶¹ Rodgers,⁶² andothers.⁶³⁻⁶⁷ Of these, only the napthophthalocyanines absorb efficientlyin the most desirable >700 nm spectral region. Unfortunately, theseparticular dyes are difficult to prepare in a chemically pure, watersoluble form and are relatively inefficient photosensitizers for singletoxygen production, perhaps even acting photodynamically via other oxygenderived toxins (e.g. superoxide). Thus a search continues for yet a"third generation" of photosensitizers which might better meet the tencritical criteria listed above.

It is an important aspect of the present invention that an improved"third generation" of photosensitizers can be obtained using large,pyrrole-containing "expanded porphyrins". These systems, beingcompletely synthetic, can, at least in principle, be tuned so as toincorporate any desired properties. Unfortunately, the chemistry of suchsystems is still in its infancy: In marked contrast to the literature ofthe porphyrins, and related tetrapyrrolic systems (e.g. phthalocyanines,chlorins, etc.), there are only a few reports of largerpyrrole-containing systems, and only a few of these meet the criterionof aromaticity deemed essential for long-wavelength absorption andsinglet oxygen photosensitization.⁶⁸ Indeed, to date, in addition to thepresent inventors' studies of texaphrin 5_(D),⁶⁹ (see FIG. 24A, FIG.24B, FIG. 24C and FIG. 24D) and "sapphyrin" 6_(D), first produced by thegroups of Woodward⁷⁰ and Johnson⁷¹, there appear to be only two largeporphyrin-like systems which might have utility as photosensitizers.These are the "platyrins" of LeGoff (exemplified by [22]platyrin7_(D))⁷² and the vinylogous porphyrins of Franck (represented by[26]porphyrin 8_(D)).⁷³ Unfortunately, to date, little has beenpublished on the photodynamic aspects of these materials, althoughcomments have been included in the most recent synthetic reports whichsuggest that such studies are in progress. The present studies, however,of expanded porphyrins 5_(D) and 6_(D), indicate that an expandedporphyrin approach to photodynamic therapy is potentially quitepromising. Interestingly, the porphycenes⁷⁴ (e.g. 9_(D)), a novel classof "contracted porphyrins" also show substantial promise as potentialphotosensitizers.⁷⁵

The present invention involves a major breakthrough in the area ofligand design and synthesis: synthesis the first rationally designedaromatic pentadentate macrocyclic ligand, thetripyrroledimethine-derived "expanded porphyrin"5_(D).⁶⁹ This compound,to which the trivial name "texaphyrin" has been assigned, is capable ofexisting in both its free-base form and of supporting the formation ofhydrolytically stable 1:1 complexes with a variety of metal cations,including a number, such as Cd²⁺, Hg²⁺, In³⁺, Y³⁺, Nd³⁺, Eu³⁺, Sm³⁺, andGd³⁺, that is too large to be accommodated in a stable fashion withinthe 20% smaller tetradentate binding core of the well-studiedporphyrins. In addition, since the free-base form of 5_(D) is amonoanionic ligand, the texaphyrin complexes formed from divalent andtrivalent metal cations remain positively charged at neutral pH. As aresult, many of these complexes are quite water soluble--at least farmore so than the analogous porphyrin complexes.

To date, two X-ray crystal structures of two different Cd²⁺ adducts havebeen obtained, one of the coordinatively saturated, pentagonalbipyramidal bispyridine complex;^(69a) the other of a coordinativelyunsaturated pentagonal pyramidal benzimidazole complex.^(69b)Importantly, both confirm the planar pentadentate structure of this newligand system and support the assignment of this prototypical "expandedporphyrin" as aromatic.

Further support for the aromatic formulation comes from the opticalproperties of 5_(D). For instance, the lowest energy Q-type band of thestructurally characterized bispyridine cadmium(II) complex of 5_(D) at767 nm (*I704*12*e*I704*10*=51,900) CHCl₃ is both considerably moreintense (by roughly a factor of 10!) and substantially red shifted (byalmost 200 nm!) as compared to that of a typical reference cadmium(II)prophyrin. Of further interest is the fact that compound 5_(D) and bothits zinc(II) and cadmium(II) complexes are very effectivephotosensitizers for singlet oxygen, giving quantum yields for ¹ O₂formation of between 60 and 70% when irradiated at 354 nm inair-saturated methanol.^(69c) It is these latter remarkable propertieswhich make these systems potentially ideal candidates for use inphotodynamic therapy and blood purification protocols.

A variety of new aromatic tripyrroledlmethine-derived macrocyclicligands analogous to compound 5_(D) above now been prepared and more areplanned. A number of these, e.g. 10_(D) -15_(D) (See FIG. 25A, FIG. 25B,FIG. 25C and FIG. 25D) have already been synthesized and found to formmetal complexes as texaphrin 5_(D) and many others can easily beconceived. This aspect of the present invention involves preparation ofnew analogues of the original texaphrin and elucidation of theirchemical and photobiological properties. Of importance is the fact that,by making ostensibly minor substitutions, one may "tune" at will theenergy of the lowest Q-type band. For instance in the sequence ofcadmium(II) complexes derived from 14_(D), 5_(D) and 16_(D) (which havealready been examined), this transition ranges from 690 to 880 nm! Thus,it appears at present, as if the optical properties of thetexaphyrin-type expanded porphyrins can be matched to any desired laserfrequency. Again, this is a feature that suggests that this class ofdyes will be well-suited for a variety of photodynamic applications.

Several preliminary in vitro biological studies have been carried-outwith the cadmium(II) complexes of the 18 and 22 π-electron texaphyrins14_(D) and 5_(D). These results, although limited in scope, areencouraging. For instance, both complexes effect a ca. 2 logphoto-killing of HSV-1 infectivity upon irradiation with 20 J/cm² oflight at the lowest energy absorption (690 nm and 767 nm, respectively),yet, importantly, neither 5_(D) nor 14_(D) show any appreciable darkantiviral activity (nor, fortunately, do they show much evidence ofgeneral cytotoxicity in the absence of light). In addition, the 22π-electron cadmium-containing texaphyrin 5_(D) has been shown by bothabsorption and emission measurements to localize selectively onlymphocytes. This latter result, in particular, augurs well for theeventual use of these materials in prophylactic photodynamic anti-AIDSblood-treatment programs. The 2 log decrease in HSV-1 activity achievedwith the texaphyrin systems studied to date is not yet sufficient tocompletely design a viable protocol: Both HPD and PII, as well assapphyrin (6_(D)), reprepared by literature methods⁷⁰ provide about a 5log decrease in viral activity under similar light fluence whenirradiated at the appropriate lowest energy transition (630 and 690 nm,respectively). Although a mechanistic comparison with the incompletelycharacterized hematoporphyrin-derived systems is difficult, a directstructural correspondence exists between the tripyrroledimethine-derivedcadmium(II) texaphyrin and free-base sapphyrin systems: The maindifference is in overall charge on the photosensitiser. It may thus bethat these two macrocycle types are bound to the virus envelope in adifferent manner; perhaps the sapphyrin "intercalates" into the lipidlayer and the charged metallotexaphyrin sits on the surface of themembrane and as such could suffer from deleterious aggregations (whichwould lower singlet oxygen production). The critical observationaldifference between these two closely related systems (texaphyrin vs.sapphyrin) suggests that small structural differences may be reflectedin significant functional effects. In addition, these experimentalfindings suggest that: 1) The free-base texaphyrin system should be afar more efficient photosensitizer for in vitro and in vivo applicationsthat the cadmium complexes studied so far, and that 2) adjusting thesubstituents on the texaphyrin periphery should serve to alter the keybiodistribution properties of the metalated and metal-free systems. Evenif all attempts to augment the photodynamic antiviral efficiency oftexaphyrin meet with failure (a result we consider highly unlikely), itis probable that this new photosensitizer will find applications in moreclassic tumor-treating procedures: The 18 π-electron cadmium-containingmacrocyclic system 14_(D), for instance, has already been shown toeffect a roughly 4 log photo-kill of Daudi-strain leukemic cells.

The synthesis of texaphyrin 5_(D) is summarized in FIG. 26. It involvedthree major steps. The first is the synthesis of the tripyrrane 18_(D).This crucial intermediate is obtained directly as the result of thesimple acid-catalyzed condensation between pyrroles 16_(D) and 17_(D).Following deprotection and formylation the key diformyl tripyrraneprecursor 20_(D) is obtained in yields exceeding 80% based on 16_(D).Condensation of this tripyrrane with O-phenylenediamine constitutes thesecond critical step in the synthetic pathway. Fortunately, thisreaction proceeds in virtually quantitative yield and gives the sp³hybridized form of the "texaphyrin" skeleton, 21_(D), directly.⁷⁶ Thefinal critical step then involves oxidation and, as appropriate,concurrent metal binding. In the case of Cd²⁺, Hg²⁺, and Zn²⁺, thearomatic, sp² hybridized, form of the macrocycle (5) is obtained inroughly 25% yield by simply stirring the starting sp³ hybridizedprecursor (21) with the appropriate salt in the presence of oxygen.⁶⁹Such a simple metal insertion and oxidation procedure, however, failsfor cations of the lanthanide series. Here, a combination of metal salt,proton sponge® (N,N',N",N'"-tetramethyl-1,8-diaminonaphthalene), andoxygen are required to effect oxidation and metal insertion.Interestingly, the use of a proton sponge alone gave the free-base formof the ligand directly, but unfortunately in only ca. 10% yield. Effortsto optimize this latter yield are still in progress.

By using a variety of other substituted diamines, it has already provedpossible to generate a range of other tripyrroledimethine-derivedmacrocycles, and it should prove possible to prepare many more by usingthe appropriate diamine and/or diformyl tripyrrane, to generate themodified texaphyrins 22_(D) -28_(D) shown in FIG. 27A, FIG. 27B, FIG.27C and FIG. 27D. In all cases, the synthesis is expected to bestraightforward. In addition to the normal condensation and oxidationsteps discussed above, the only new transformations required willinvolve exposure to basic reagents (i.e. to effect saponification of anester) under conditions where the macrocyclic skeleton itself is knownto be stable. It should also be noted that all of the necessaryprecursors, with the exception of that required for 27_(D), are eitheravailable commercially or already on hand.

A further advantage of developing a wide variety of solubilizedtexaphyrins is that many of these would be suitable for furtherfunctionalization. For instance, treatment of texaphyrins 25_(D) or26_(D) with thionyl chloride or p-nitrophenol acetate would generateactivated acyl species suitable for attachment to monoclonal antibodiesor other biomolecules of interest. Alternatively, standard in situcoupling methods (e.g. DCCI) could be used to effect the same sort ofconjugation. In either case, the ability to attach and deliver a potentphotosensitizer directly to a tumor locus could have tremendouspotential benefit in the treatment of neoplastic disorders.⁷⁷

All of the texaphyrin systems prepared to date, and all the new targetsystems proposed above, involve an imine-containing macrocyclic core.The use of such a linking group offers both advantages anddisadvantages. The primary advantage is that macrocyclic systemscontaining such subunits are easy to prepare and generally act aseffective ligands (this is certainly true for texaphyrin!). On the otherhand, they are thermodynamically unstable with regards to hydrolysis,although, at least in the case of texaphyrin 5_(D), this is less of aproblem than one might expect. For instance, the half-life for iminehydrolysis of the best-studied cadmium-containing complex of 5_(D) isroughly 30 days at pH 7 and several hours at pH 2. Nonetheless,applications could be envisioned where greater stability might berequired. For this reason the synthesis of the two methine-linkedtexaphyrin analogues 29_(D) and 30_(D) in which the weakest CH═N linkhas been replaced by a more robust CH═CH subunit (See FIG. 28A and FIG.28B) is an aim of this invention. It is an expectation that compounds29_(D) and 30_(D) may be prepared using either standard Wittig-basedring closures, which have proved useful in the synthesis of large,furan-containing annulenes,⁷⁸ or via McMurry-type couplings, such asthose that have recently proved useful in the synthesis ofporphycenes.⁷⁴

Once in hand, all new texaphyrin systems will be characterized fullyusing normal spectroscopic and analytical means, including, wherepossible X-ray diffraction methods. In addition, a complete analysis ofthe optical properties will be made for all new systems under a range ofexperimental conditions including some designed to approximate thosewhich might pertain in vivo. Initial measurements such as simplerecording of the optical absorption and emission spectra will be carriedout in the P.I.'s laboratory. More detailed analyses, including tripletlifetime and singlet oxygen quantum yield determinations, will becarried out. The objective of this part of the proposed research programis to obtain a complete ground and excited state reactivity profile foreach and every new texaphyrin produced. Questions such as when issinglet oxygen production maximized, how is the quantum yield for itsformation influenced by the position of the lowest energy (Q-type)transition, whether aggregation is more prevalent in certain solvents orin the presence of certain biologically important components (e.g.lipids, proteins, etc.), and, finally, whether significant differencesin in vitro optical properties are derived from the use of elaboratedtexaphyrins bearing cationic, anionic, or neutral substituents all willbe thus answered.

Once the above complexes are made, screening experiments are carriedout. Standard in vitro protocols will be used to evaluate the in vitrophoto-killing ability of the texaphyrin derivatives in question. Forinstance, the dyes of choice will be administered in varyingconcentrations to a variety of cancerous cells and the rate ofreplication determined both in the presence and absence of light.Similarly, dyes of choice will be added to standard vital cultures andthe rate of growth retardation determined in the presence and absence oflight. Where appropriate, a variety of solubilizing carriers will beused to augment the solubility and/or monomeric nature of the texaphyrinphotosensitizers and the effect, if any, that these carriers have inadjusting the biodistribution properties of the dyes will be assessed(using primarily fluorescence spectroscopy). It should be stressed, ofcourse, that in all cases appropriate control experiments will becarried out with normal cells so that the intrinsic dark and lighttoxicity of the texaphyrins may be determined.

From a generalized set of in vitro experimental procedures, it isexpected that a clear picture of the photodynamic capabilities of thetexaphyrin system will emerge. Again, as above, key questions aboutstructure and reactivity will be addressed and answered in what is(hopefully) an unambiguous fashion. In addition, some preliminarytoxicity and stability information will begin to emerge from these invitro experiments. Here questions of interest include how long thetexaphyrin system is holding up under physiological conditions andwhether the nature of the central metal influences this stability.Equally, or perhaps more important is the question of whether thecentral cation is affecting cytotoxicity. As discussed in paperspublished by the present inventors,^(69b),69d it is not possible toremove the larger bound cations (e.g. Cd²⁺ or Gd³⁺) by simple chemicalmeans (Zn²⁺, however, appears to "fall out" with ease). Moreover,preliminary results suggest that the best-studied cadmium(II)-containingtexaphyrin complex 5_(D) is not appreciable cytotoxic. Nonetheless, thequestion of intrinsic toxicity is one of such central importance thatthe cytotoxicity of all new systems will be screened in vitro and, wheredeemed appropriate, further in vivo toxicity studies will also becarried out.

Once in vitro screening experiments are complete, samples of potentialphotosensitizers that look particularly promising will be selected forfurther development. Those that possess the best combination ofstability and photodynamic ability for use in blood treatment protocolswill be further evaluated in flow system using whole blood samples.Those that look promising for tumor treatment will be subjected tofurther animal screening.

This aspect of the present invention involves the coordination andphotochemical properties of tripyrroledimethine-derived "texaphyrins", anew class of "expanded prophyrins", the first members of which have beenrecently prepared and characterized in our laboratory. It is expectedthat these basic studies will lead to the development of viableprocedures of removing HIV-1 and other enveloped viruses from transfusedblood as well as improved means of detecting and treating tumors. Thelong range goals exemplified here are to:

1. Synthesize further safe and efficient photosensitizers for use inkilling human immunodeficiency virus (HIV-1) and other enveloped virusesin blood, which operate without harm to normal blood components.

2. Develop new safe and effective photosensitizers for use in the invivo photodynamic treatment of tumors.

The approach to these long range objectives centers around thepreparation and use of suitably modified tripyrrole-dimethine derivedtexaphyrin-type expanded porphyrins. This is an essential first steptowards the realization of the above goals. Specific extensions of thepresent invention include:

1. Explore further the coordination and general chemical properties ofour original texaphyrin and existing analogues and obtain a completesolubility, stability, and reactivity profile for those complexes deemedlikely to be of greatest biomedical interest.

2. Synthesize simple analogues of the currently available texaphyrinswith cationic, anionic, or neutral substituents and study how suchmodifications alter the water solubility and biodistribution propertiesof these expanded porphyrins.

3. Make analogues of texaphyrin which contain reactive nucleophilic orelectrophilic substituents suitable for conjugation to monoclonalantibodies or other biomolecules of potential interest.

4. Prepare new texaphyrin-type aromatic macrocycles in which the keyimine (CH═N) functionality has been replaced with a presumably morerobust methine (CH═CH) linkage.

5. Carry out complete photochemical studies of all new texaphyrins so asto determine unambiguously those factors (e.g. λ_(max)) which maximizesinglet oxygen production.

6. Test the in vitro photodynamic tumor and virus killing efficiency ofthe new texaphyrins prepared in the course of the synthetic phase ofthis project.

7. Test the in vivo photodynamic anti-tumor properties of the morepromising texaphyrins synthesized and screened as outlined above.

Literature citations in the following list are incorporated by referenceherein for the reasons cited.

REFERENCES

1. Confronting AIDS, National Academy of Sciences Press: Washington,D.C., 1988.

2. Dougherty, T. J.; Kaufman, J. E.; Goldfarg, A.; Weishaupt, K. R.;Boyle, D.; Mittleman, A. Cancer Res. 1978, 38, 2628.

3. Dahlman, A.; Wile, A. G.; Burns, R. G.; Mason, G. R.; Johnson, F. M.;Berns, M. W. Cancer Res. 1983, 43, 430.

4. Dougherty, T. J. in Methods in Porphyrin Photosensitization, Kessel,D., Ed.; Plenum Press: New York, 1985; pp. 313-328.

5. Dougherty, T. J. Photochem. Photobiol. 1987, 45, 879.

6. See for instance: (a) Figge, F. H. J.; Weiland, G. S. Anat. Rec.1948, 100, 659; (b) Rasmussen-Taxdall, D. S.; Ward, G. E.; Figge, F. H.Cancer (Phila.) 1955, 8, 78.

7. Berenbaum, M. C.; Bonnett, R.; Scourides, P. A. Br. J. Cancer 1982,47, 571.

8. Berns, W.; Dahlman, A; Johnson, F. M.; et al. Cancer Res. 1982, 42,2326.

9. Dougherty, T. J.; Gomer, C. J.; Weishaupt, K. R. Cancer Res. 1976,36, 2330.

10. Dougherty, T. J. Photochem. Photobiol. 1983, 38, 377.

11. Evensen, J. F.; Sommer, S.; Moan, J.; Chistensen, T. Cancer Res.1984, 44, 482.

12. Gibson, S. L. Hilf, R. Photochem. Photobiol. 1985, 42, 367.

13. Gomer, C. J.; Smith, D. M. Photochem. Photobiol. 1980, 32, 341.

14. Herra-Ornelas, L.; Petrelli, N. J.; Mittleman, A.; Dougherty, T. J.;Boyle, D. G. Cancer, 1986, 57, 677.

15. Kessel, D. Photochem. Photobiol. 1984, 39, 851.

16. Kessel, D. Photochem. Photobiol. 1986, 44, 489.

17. Kessel, D. Int. J. Radiat. Biol. 1986, 49, 901.

18. Klaunig, J. E.; Selman, S. H.; Shulok, J. R.; Schaefer, P. J.;Britton, S. L.; Goldblatt, P. J. Am. J. Path. 1985, 119, 230.

19. Moan, J.; Somer, S. Cancer Lett. 1987, 21, 167.

20. Moan, J.; Peng, Q.; Evensen, J. F.; Berg, K.; Western, A.;Rimington, C. Photochem. Photobiol. 1987, 46, 713.

21. Singh, G.; Jeeves, W. P.; Wilson, B. C.; Jang, D. Photochem.Photobiol. 1987, 46, 645.

22. Bonnett, R.; Ridge, R. J.; Scourides, P. A. J. Chem. Soc., PerkinTrans. I 1981, 3135.

23. Chang, C. K.; Takamura, S.; Musselman, B. D.; Kessel, D. ACS Adv.Chem. Ser. 1986, 321, 347.

24. Dougherty, T. J. Photochem. Photobiol. 1987, 46, 569.

25. Kessel, D. Photochem. Photobiol. 1986, 44, 193.

26. Moan, J.; Christensen, T.; Somer, S. Cancer Lett. 1982, 15, 161.

27. Scourides, P. A.; Bohmer, R. M.; Kaye, A. H.; Morstyn, G. CancerRes. 1987, 47, 3439.

28. Blum, A.; Grossweiner, L. I. Photochem. Photbiol. 1985, 41, 27.

29. Henderson, B. W.; Miller, A. C. Radiat. Res. 1986, 108, 196.

30. Keene, J. P.; Kessel, D.; Land, E. J.; Redmond, R. W.; Truscott, T.G. Photochem. Photobiol. 1986, 43, 117.

31. Parker, J. G. Lasers Surg. Med. 1986, 6, 258.

32. Tanielian, C.; Heinrich, G.; Entezami, A. J. Chem. Soc., Chem.Commun. 1988, 1197.

33. Weishaupt, K. R.; Gomer, L. J.; Dougherty, T. J. Cancer Res. 1976,36, 2326.

34. Gulliya, K. S.; Matthews, J. L.; Fay, J. W.; Dowben, R. M. LifeSciences 1988, 42, 2651.

35. Matthews, J. L.; Newman, J. T.; Sogandares-Bernal, F.; Judy, M. M.;Kiles, H.; Leveson, J. E.; Marengo-Rowe, A. J.; Chanh, T. C.Transfusion, 1988, 28, 81.

36. Skiles, H.; Sogandares-Bernal, F.; Judy, M. M.; Matthews, J. L.;Newman, J. T. Abstracts of 6th Southern Biomedical EngineeringConference, 1987, 83.

37. Skiles, H.; Judy, M. M. Newman, J. T. in Abstracts of the AnnualMeeting of the American Society for Microbiology, 85th Annual Meeting,Mar. 3-7, 1985, p. 7, A 38.

38. Lewin, A. A.; Schnipper, L. E.; Crumpacker, C. S. Proc. Soc. Exptl.Biol. Med. 1980, 163, 81.

39. Schnipper, L. E.; Lewin, A. A.; Swartz, M.; Crumpacker, C. S. J.Clin. Invest. 1980, 65, 432.

40. Curran, J. W.; Lawrence, D. N.; Jaffe, H.; et al. N. Engl. J. Med.1984, 310, 69.

41. Groopman, J. E.; Hartzband, P. I.; Shulman, L.; et al. Blood 1985,66, 742.

42. Ward, J. W.; Deppe, D. A.; Samson, S.; et al. Ann. Intern. Med.1987, 106, 61.

43. Ward, J. W.; Holmber, S. D.; Allen, J. R.; et al. N. Engl. J. Med.1988, 318, 473.

44. Ho, D. D.; Pomerantz, R. J.; Kaplan, J. C. New Engl. J. Med. 1987,317, 278.

45. Christensen, T.; Sandquist, T.; Feven, K.; Waksvik, H.; Moan, J. Br.J. Cancer 1983, 48, 35.

46. Profio, A. E.; Doiron, D. R. Photochem. Photobiol. 1987, 46, 591.

47. Wan, S.; Parrish, J. A.; Anderson, R. R.; Madden, M. Photochem.Photobiol 1981, 34, 679.

48. Eichler, J.; Knop, J.; Lenz, H. Rad. Environ. Biophys. 1977, 14,239.

49. Oseroff, A. R.; Ohuoha, D.; Ara, G.; McAuliffe, D.; Foley, Jr.;Cincotta, L. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 9729; andreferences therein.

50. Gulliya, K. S.; Matthews, J. L. Cell Biol. Int. Rep. 1988, 12, 305;and references therein.

51. Detty, M. R.; Merkel, P. B.; Powers, S. K. J. Am. Chem. Soc. 1988,110, 5920.

52. Berenbaum, M. C.; Akande, S. L.; Bonnett, R.; Kaur, H.; Ioannou, S.;White, R. D.; Winfield, U.-J. Br. J. Cancer 1986, 54, 717.

53. Kessel, D.; Thompson, P.; Saatio, K.; Nanwi, K. D. Photochem.Photobiol. 1987, 45, 787.

54. Bonnett, R.; McGarvey, D. J.; Harriman, A.; Land, E. J.; Truscott,T. G.; Winfield, U.-J. Photchem. Photobiol. 1988, 48, 271.

55. (a) Morgan, A. R.; Tertel, N. C. J. Org. Chem. 1986, 51, 1347;Morgan, A. R.; Garbo, G. M.; Kreimer-Birnbaum, M.; Keck, R. W.;Chaudhuri, K.; Selman, S. H. Cancer Res. 1987, 47, 496.

56. Morgan, A. R.; Rampersaud, A.; Keck, R. W.; Selman, S. H. Photochem.Photobiol. 1987, 46, 441.

57. Beems, E. M.; Dubbelman, T. M. A. R.; Lugtenburg, J.; Van Best, J.A.; Smeets, M. F. M. A.; Boeheim, J. P. J. Photochem. Photobiol. 1987,46, 639.

58. Cubeddu, R. Keir, W. F.; Ramponi, R.; Truscott, T. G. Photochem.Photobiol. 1987, 46, 633.

59. (a) Kessel, D.; Dutton, C. J. Photochem. Photobiol. 1984, 40, 403;(b) Kessel, D. Cancer Res. 1986, 46, 2248.

60. (a) Dolphin, D. 196th American Chemical Society Meeting, LosAngeles, September 1988, Abstract no. 312; (b) Richter, A. M.; Kelly,B.; Chow, J.; Liu, D. J.; Towers, G. H. N.; Dolphin, D.; Levy, J. G.Cancer Res., in press.

61. (a) Ben-Hur, E.; Rosenthal, I,. Inter. J. Radiat. Biol. 1985, 47,145; (b) Ben-Hur, E.; Rosenthal, I. Photochem. Photobiol. 1985, 42, 129;(c) Ben-Hur, E.; Rosenthal, I. Rad. Res. 1985, 103, 403; (d) Selman, S.H.; Kreimer-Birnbaum, M.; Chaudhuri, K.; Garbo, G. M.; Seaman, D. A.;Keck, R. W.; Ben-Hur, E.; Rosenthal, I. J. Urol. 1986, 136, 141; (e)Ben-Hur, E.; Rosenthal, I. Cancer Lett. 1986, 30, 321; (f) Ben-Hur, E.;Rosenthal, I. Photochem. Photobiol. 1986, 43,615; (g) Ben-Hur, E.;Green, M.; Prager, A.; Kol, R.; Rosenthal, I. Photochem. Photobiol.1987, 46, 651.

62. (a) Firey, P. A.; Rodgers, M. A. J. Photochem. Photobiol. 1987, 45,535; (b) Firey, P. A.; Ford, W. E.; Sounik, J. R.; Kenney, M. E.;Rodgers, M. A. J. J. Am. Chem. Soc. 1988, 110, 7626.

63. (a) Skikes, J. D. Photochem. Photobiol. 1986, 43, 691; (b) Spikes,J. D.; Bommer, J. C. Int. J. Rad. Res. 1986, 50, 41.

64. Brasseur, N.; Ali, H. Autenrieth, D.; Langlois, R.; van Lier, J. E.Photochem. Photobiol. 1985, 42, 515.

65. Bown, S. G.; Tralau, C. J.: Coleridge Smith, P. D.; Akdemir, D.;Wieman, T. V. Br. J. Cancer 1986, 54, 43.

66. Chan, W.-S; Svensen, R.; Phillips, D.; Hart, I. R. Br. J. Cancer1986, 53, 255.

67. Sonoda, M.; Krishna, C. M.; Riesz, P. Photochem. Photobiol. 1987,46, 625.

68. For a review see: Sessler, J. L.; Cyr, M.; Murai, T. Comm. Inorg.Chem. 1988, 7, 333.

69. (a) Sessler, J. L.; Mural, T.; Lynch, V.; Cyr, M. J. Am. Chem. Soc.1988, 110, 5586; (b) Sessler, J. L.; Murai, T.; Lynch, Inorg. Chem. Inpress; (c) Harriman, T.; Maiya, B. G.; Murai, T.; Hemmi, G.; Sessler, J.L.; Mallouk, T. E. J. Chem. Soc., Chem. Commun., in press; (d) Sessler,J. L.; Murai, T.; Hemmi, G. Inorg. Chem., submitted.

70. Bauer, V. J.; Clive, D. R.; Dolphin, D.; Paine, J. B. III; Harris,F. L.; King, M. M.; Loder, J.; Wang, S.-W. C.; Woodward, R. B. J. Am.Chem. Soc. 1983, 105, 6429.

71. Broadhurst, M. J.; Grigg, R.; Johnson, A. W. J. Chem. Soc. PerkinTrans. 1, 1972, 2111.

72. (a) Berger, R. A.; LeGoff, E. Tetrahedron Lett. 1978, 4225. (b)LeGoff, E.; Weaver, O. G. J. Org. Chem. 1987, 710.

73. (a) Gosmann, M.; Franck, B. Angew. Chem. 1986, 98, 1107; Angew.Chem. Int. Ed. Eng. 1986, 25, 1100. (b) Knubel, G.; Franck, B. Angew.Chem. 1988, 100, 1203; Angew. Chem. Int. Ed. Eng. 1988, 27, 1170.

74. (a) Vogel, E.; Kocher, M.; Schmickler, H.; Lex, J. Angew. Chem.1986, 98, 262; Angew. Chem. Int. Ed. Eng. 1986, 25, 257. (b) Vogel, E.;Balci, M.; Pramod, K.; Koch, P.; Lex. J. Ermer, O. Angew. chem. 1987,99, 909; Angew. Chem. Int. Ed. Eng. 1987, 26, 928.

75. Aramendia, P. F.; Redmond, R. W.; Nonell, S.; Schuster, W.;Braslavsky, S. E.; Schaffner, K.; Vogel, E. Photochem. Photobiol. 1986,44, 555.

76. Sessler, J. L.; Johnson, M. R.; Lynch, V. J. Org. Chem. 1987, 52,4394.

77. For an example of porphyrin-antibody conjugation and a discussion ofthe relative merits of various coupling methods see, for instance:Mercer-Smith, J. A.; Roberts, J. C; Figard, S. D.; Lavallee, D. K. in"Antibody-Mediated Delivery Systems," Rodwell, J. D.; Ed. Marcel Dekker:New York; 1988, pp. 317-352.

78. Vollhardt, K. P. C. Synthesis 1975, 765.

EXAMPLE 6

One aspect of the utility of the present invention is exemplified by useof complexes described herein for photon-induced deactivation of virusesand virally infected or potentially infected encaryotic cells. Thegeneral photodeactivation method used in this example was developed bythe Infectious Disease and Advanced Laser Applications Laboratories ofthe Baylor Research Foundation, Dallas, Tex. and is a subject of a U.S.patent application filed Jun. 25, 1987 by Millard Monroe Judy, JamesLester Matthews, Joseph Thomas Newman and Franklin Sogandares-Bernal(assigned to the Baylor Research Foundation, Dallas, Tex.).

The efficiency of some of the porphyrin-like macrocycles inphotosensitized inactivation of Herpes Simplex Virus Type 1 (HSV-1) andof human lymphocytes and monocytes, both peripheral mononucleatedvascular cells (PMC) and cellular hosts of HIV-1 has been initiated.Previous studies of viral inactivation using the macrocyclicphotosensitizers dihematoporphyrin ether (DHE) or hematoporphyrinderivative (HPD) have shown that with the porphyrins, only those virusesstudied which are enveloped or possess a membraneous coat areinactivated. The enveloped viruses studied include HSV-1,cytomegalovirus, measles virus¹, and the human immunodeficiency virusHIV-1².

The photosensitized inactivation of Herpes Simplex Virus, Type 1 (HSV-1)was investigated in culture medium using various macrocycles of thepresent invention. Results are listed in Table 4.

                  TABLE 4                                                         ______________________________________                                        Herpes Simplex Virus I Inactivation with Assymmetric                          Expanded Porphyrin Macrocycle Complexes*                                                                % Survival Viral                                    Complex**     Conc. (μM)                                                                             Infectivity                                         ______________________________________                                        3.sub.A       20          12                                                                10          8                                                                 2.5         20                                                                0.25        100                                                 10.sub.D (where M = Cd)                                                                     20          4                                                                 10          14                                                                2.5         42                                                                0.25        100                                                 14.sub.D (where M--Cd)                                                                      16.0        3                                                                 4.0         50                                                                0.40        100                                                 ______________________________________                                         *All light irradiation at * max absorption and to give a light fluence of     10 J/cm.sup.2                                                                 **Structural formulas in FIG. 4 and FIG. 25.                             

The three cadmium-containing macrocycles (3_(A), 10_(D) (where M is Cd),and 14_(D) (where M is Cd)) at concentrations of 20 μM demonstrated ≧90%viral inactivation as judged by viral plaque assay.

The macrocycle photosensitizing studies employed enveloped HSV-1 as themodel for screening based on its ease of propagation and assessment ofinfectivity in cell culture. The screening procedure forphotoinactivation of HSV-1 was similar to the methods previouslydescribed.³ Essentially, selected macrocycles at differentconcentrations were added to a cell-free suspension of 10⁶ PFU/ml ofHSV-1. The viral suspensions were irradiated at the optimal absorptionwavelength of the selected dye at different light-energy densities.Controls consisted of (1) nonirradiated virus, (2) virus irradiated inthe absence of macrocycle, and (3) virus treated with selectedconcentrations of macrocycle and maintained in the dark. All sampleswere then assessed for viral infectivity by determining the number ofPFU/ml in Vero cells.

Viral suspensions were serially diluted and subsequently absorbed ontoVero cell monolayers for 11/2 hours at 37° C. An overlay medium wasadded and the cells incubated at 37° C. for 3-4 days. The overlay mediumwas then removed, the monolayers fixed with methanol and tinctured withGiemsa, and individual plaques counted under a dissecting microscope.Uninfected cell cultures also were exposed to the macrocycle complexesto rule out direct cytotoxic effects.

The inactivation rich of PMC's in the absence and presence of lightafter exposure to concentrations of complex 3_(A) in whole human plasmaranging from 0.015 to 38 μM is shown in FIG. 29 and FIG. 30.Inactivation was judged by mitogenic assay. Toxicity onset with 3_(A)(see FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D) and 1_(C) (see FIG. 17) inthe absence of light was between 0.15 and 1.5 μM (FIG. 29). As shown bymitogenic assay in FIG. 30, aerobic photosensitization of cells exposedto 3_(A) at 0.15 μM concentration and 20 joules/cm² of 770 nm wavelengthlight caused significant inhibition of the cellular division of PMC's.Moderate increase in either photosensitizer concentration or lightdosage is expected to result in sequentially complete cellularinactivation.

The results to date, some of which are summarized herein, indicatestrongly that the expanded porphyrin-like macrocycles of the presentinvention should be efficient photosensitizers for free HIV-1 andinfected mononuclear cells as well. Altering the polarity and electricalcharges of side groups of these macrocycles is anticipated to altermarkedly the degree, rate, and perhaps site(s) of binding to freeenveloped viruses such as HIV-1 and to virally-infected peripheralmononuclear cells. These substituent changes are also expected tomodulate photosensitizer take-up and photosensitization of leukemia orlymphoma cells contaminating bone-marrow as well as by normal cells ofthe marrow.

References in the following literature incorporated by reference hereinfor the reasons cited.

REFERENCES

1. Skiles, H. L., Judy, M. M. and Newman, J. T. Abstracts to the AnnualMeeting of the ASM, A38, pg. 7, 1985.

2. Matthews, J. L., Newman, J. T., Songandares-Bernal, F., Judy, M. M.,Skiles, H., Leveson, J. E., Marengo-Rowe, A. J., and Chanh, T. C.Transfusion, 28:81, 1988.

3. Skiles, H. F., Sogandares-Bernal, F., Judy, M. M., Matthews, J. L.and Newman, J. T. Biomedical Engineering VI: Recent developments. SixthSouthern Biomedical Engineering Conference, 1987.

EXAMPLE 7

This example summarizes certain of the basic five-coordinate(pentadentate), expanded porphyrin compounds and complexes of thepresent invention and their derivatives which have been synthesised. InFIG. 31A, FIG. 31B and FIG. 31C, compounds 1_(B) -7_(B), 14_(D) and15_(D) are shown. Variations include changes in theortho-phenylenediamino substituent, namely R₁ and R₂ and in the natureof the starting diamine itself. When both R¹ and R² on the orthophenylene diamino substituent are hydrogen as in compound 1_(B) thebasic structure of texaphyrin is shown. These R¹ and R² substituents mayalso both be methyl, CH₃ (compound 2_(B)). Additionally when R¹ is H, R²may be chloride (compound 3_(B)); bromine (compound 4_(H)); nitro(compound 5_(B)); methoxy (compound 6_(B)); or carboxy (compound 7_(B)).When M is hydrogen, the complex has a charge of 0 (n=0). When M is adivalent metal such as mercury⁺² , cadmium⁺², zinc⁺², cobalt⁺² ormanganese⁺² ; the charge of the complex will be +1 (n=1). When M is atrivalent metal cation such as europium⁺³, neodynium⁺³, samarium⁺³,lanthanum⁺³, gadolinium⁺³, indium⁺³ or yttrium⁺³ the charge of thecomplex will be +2 (n=2). For the complexes marked with a singleasterisk (1_(B) and 2_(B)) all metals, divalent or trivalent mentionedabove have been included in various formed complexes. Complexes with adouble asterisk (3_(B) -6_(B)) have been synthesized as either the zincor cadmium derivative (M=Zn or Cd; n=1). While many other usablecompounds are mentioned in other sections of this application, or couldbe readily synthesized by those of skill in the art from the directionsincluded herein, the particular ones mentioned in this Example arethought to be especially useful for many purposes, for example, thoseinvolving purifying biological samples of viruses, particularlyretroviruses. These compounds should also be useful for processes suchas, for example, photodynamic cancer therapy, magnetic resonance imaging(MRI) enhancement and antibody functionalization as mentioned elsewhereherein.

EXAMPLE 8 Magnetic Resonance Imaging Enhancement

In many respects the key to cancer control lies as much, if not more, inearly detection and diagnosis as it does in subsequent therapeuticmanagement. New techniques which allow neoplastic tissue to be observedand recognized at an early stage of development thus have a criticalrole to play in the battle against these disorders. One such promisingtechnique is magnetic resonance imaging (MRI). ¹⁻⁵ Although quite new,this noninvasive, apparently innocuous method, is not firmly entrenchedas a diagnostic tool of prime importance, complementing or, in somecases, supplanting computer assisted X-ray tomography as the method ofchoice for solid tumor detection.

The physical basis of current MRI methods has its origin in the factthat in a strong magnetic field the nuclear spins of water protons indifferent tissues relax back to equilibrium at different rates, whensubject to perturbation from the resting Boltzman distribution by theapplication of a short rf pulse.²,5 For the most common type ofspin-echo imaging, return to equilibrium takes place in accord withequation 1 and is governed by two time constants T₁ and T₂, thelongitudinal and transverse relaxation times, respectively.

    SI=[H]H(ν){esp(-T.sub.E /T.sub.2)}{1-exp (-T.sub.R /T.sub.1)}(1)

Here, SI represents the signal intensity, [H] is the concentration ofwater protons in some arbitrary volume element (termed a voxel), H(ν) amotion factor corresponding to motion (if any) in and out of this volumeelement, and T_(E) and T_(R) are the echo-delay time and thepulse-repetition times, respectively. The various pulse sequencesassociated with obtaining an MRI image thus correspond to choosing T_(E)and T_(R) by setting the times associated with (and between) theexcitation and interrogation rf pulses (the first to perturb the system,the second to determine the extent of return to equilibrium) anddetermining SI, which, as illustrated above, is function of theparticular T₁ and T₂ values in force. Since both T₁ and T₂ are afunction of the local (bulk) magnetic environment, and as such are afunction of the particular tissue in which the water proton is situated,differences in these values (and hence SI) allow for imagereconstruction. Of course, only when these local, tissue-dependent,relaxation differences are large can tissue differentiation be effected.

In practice for biological systems, T₂ values are very short (and T_(E)and T_(R) are chosen to accentuate this situation). Thus it isdifferences in the longitudinal time constant (T₁) which dominaterelaxation effects and relative signal intensity: Decreases in T₁correspond to increasing signal intensity. Any factors, therefore, whichwill serve to decrease T₁ selectively for a particular tissue or organwill thus lead to increased intensity for that area and better contrast(signal to noise) relative to the bulk animal background. This is whereparamagnetic MRI contrast agents come into play.⁴,5

It has been known since the earliest days of magnetic resonancespectroscopy that paramagnetic compounds, containing one or moreunpaired spins, enhance the relaxation rates for the water protons inwhich they are dissolved.⁶ The extent of this enhancement, termedrelaxivity, is, in the absence of colligative interactions, given byR_(i) (in units of M-¹ s-¹ or mM-¹ s-¹) in eq. 2.⁴,5

    (1/T.sub.i).sub.obsd =(1/T.sub.i).sub.d +R.sub.i [M] i=1,2 (2)

Here, (1/T_(i))_(obsd) is the reciprocal of the observed relaxation timein the presence of a paramagnetic species M, and (1/T_(i))_(d) is theobserved relaxations time in its absence. The relaxivity of any givenparamagnetic species, i.e., metal complexes for MRI enhancement, isdependent on the magnitude of the dipole-dipole interactions between theelectron spin (on the metal) and the proton spin (on the water). Theextent of this interaction is strongly dependent on the nature of theinteraction between the paramagnetic complex and the water molecule inquestion. Traditionally, it has proved convenient to define both "innersphere" and "outer sphere" contributions to the total relaxivityR_(i).⁴,5 The former account for water molecules which participatedirectly in the coordination sphere of the metal; the latter for allother loose interactions (e.g. hydrogen bonding and translationaldiffusion of waters bound in the second coordination sphere). Wherechemically viable, it is, in general, inner sphere relaxation whichdominates R_(i). For this interaction, the contribution to thelongitudinal relaxation is given by equation 3.⁴,5

    (1/T.sub.1).sub.(inner sphere) =P.sub.M·q /T.sub.1M +τ.sub.M)(3)

Here, P_(M) is the mole fraction of metal ion, q is the number of boundwater molecules, τ_(M) is the lifetime of the bound water, and T_(1M) isthe relaxation time of the bound water protons. The value of this latterterm is approximated by the Solomon-Bloembergen equations (eqs. 4-6)which account for both dipole-dipole ("through space") and contact("through-bond") terms.⁷ ##EQU1## Here, γ₁ is the proton gyromagneticratio, g the electronic g-factor, S the total electron spin of theparamagnetic ion, β the Bohr magneton, r the water proton-metal iondistance, [A2π/h] the electron-nuclear hyperfine coupling constant, andω_(s) and ω_(I) the electronic and proton Larmor precession frequencies,respectively. The dipolar and scaler correlation times τ_(C) and τ_(e)are given by:

    1/τ.sub.C =1/T.sub.le +1/τ.sub.M +1/τ.sub.R    (5)

    1/τ.sub.e =1/T.sub.le +1/τ.sub.M                   (6)

where, T_(le) is the longitudinal electron spin relaxation time andτ_(R) the rotational tumbling time of the entire water-complex ensemble.More elaborate theoretical treatments are available to account forcollisional relaxation effects and other factors which would perturb thestatic zero-field splitting of the electronic sublevels in inner sphererelaxation pathways.⁵ Detailed analyses are also available to accountfor contributions from outer sphere mechanisms.⁵ Nonetheless, for thesake of the present discussion, the simple Solomon-Bloembergen equationsgiven above will suffice: They illustrate the key physical featuresrequired for a good paramagnetic contrast agent.

From a physical point of view, MRI contrast agents require species thatare highly paramagnetic (so that the magnetic moment term S(S+1) islarge), possess large T_(le) 's, and display large rotational tumblingtimes (τ_(R)). In addition, an ideal contrast agent should also bind oneor more water molecules (so that the inner sphere relaxation mechanismsare operative) and exchange these waters at a rate (1/τ_(M)) that isoptimal.⁴,5,8,9 with the exception of τ_(R), which is often set more bythe effective viscosity of the local environment (i.e. is the complex"stuck" to a slowly rotating protein?¹⁰) than by the choice of complex,all of these factors may be influenced by the choice of basicparamagnetic cation and by subsequent ligand design.⁴,5,9 This liganddesign, which constitutes a major thrust in current MRI research, is, ofcourse, also subject to very stringent biological requirements: Not onlymust the putative contrast agent be highly paramagnetic and achieve goodrelaxation enhancement, it must also be nontoxic at the dosagesadministered, stable in vivo, excreted quickly after diagnosis iscomplete, and, of course, show desirable tissue localizing abilities.Taken together these criteria are quite severe.

Indeed, at present, only one paramagnetic MRI contrast agent is inclinical use, the bis(N-methyl-glucamine) salt of Gd(III)diethylenetriaminepentaacetate, (MEG)₂ [Gd(DTPA)(H₂ O)] (c.f. structure10)¹¹⁻¹⁸ marketed by Berlex Laboratories. This dianionic complexlocalizes selectively in extracellular regions, and is being usedprimarily in the visualization of the capillary lesions associated withcerebral tumors.¹¹⁻¹³ In [Gd(DTPA)(H₂ O)]²⁻, one molecule of water isbound in the first (inner) coordination sphere and at 37° C. in waterthis complex displays a relaxivity of 3.7 mM-¹ s-¹ at 20 MHz.⁴,9,19 Inmarked contrast to the simple Gd(III) complex of EDTA, for which logK_(assc). =17.4 at 25° C.,²⁰,21 the DTPA complex appears to besufficiently thermodynamically stable (log K_(assc). =22.5 at 25°C.²⁰,21 so as to be kinetically stable under physiological conditionsand is apparently excreted intact through the kidneys within severaldays of administration.¹⁴ These desirable features notwithstanding, itis clear that other contrast agents with superior kinetic stability,better relaxivity, lower net charge (which would lower the osmolalityand hence pain threshold of the administered solutions), and/ordifferent tissue localizing capabilities would be desirable for clinicaluse. Indeed, in a recent review on the subject,⁵ Lauffer states that:"New synthetic methods toward kinetically inert complexes, especiallythose of Gd(III), need to be developed. These preferably should beversatile enough to allow for specific substitutions on the complex thatmay modulate its properties."

To date, in fact, considerable effort has been devoted to thedevelopment of new potential MRI contrast agents.²¹⁻³⁷ Most of this workhas centered around preparing new complexes of Gd(III).²¹⁻²⁹,362,376 Theemphasis on Gd(III) salts stems from the fact that this cation, with 7unpaired f-electrons, has a higher magnetic moment than otherparamagnetic cations such as Fe(III) and Mn(II).⁴,5 Thus, all otherthings being equal, complexes of Gd(III) would be expected to besuperior relaxation agents than those derived from Mn(II) or Fe(III). Inaddition, both iron and, to a lesser extent, manganese are sequesteredand stored very efficiently in humans (and many other organisms) by avariety of specialized metal-binding systems.³⁸ Moreover both iron andmanganese are capable of existing in a range of oxidation states and areknown to catalyze a variety of deleterious Penton-type free-radicalreactions.³⁹ Gadolinium(III), which suffers from neither of thesedeficiencies thus appears to offer many advantages. Unfortunately, as istrue for Fe(III) and Mn(II), the aqueous solution of Gd(III) is tootoxic to be used directly MRI imaging at the 0.01 to 1 mM concentrationsrequired for effective enhancement.⁴,5 Hence the emphasis is ondeveloping new agents which, as is true for DTPA, form hydrolyticallystable complexes in vivo with Gd(III) and/or other paramagnetic cations.A number of such ligands, including the very promising DOTA²¹⁻²⁷ andEHPG²⁸,29 systems, are now known (c.f. reference 5 for an extensivereview). In almost all cases, however, reliance is made on the samebasic philosophical approach. Specifically, for Gd(III) binding,carboxylates, phenolates, and/or other anionic chelating groups arebeing used to generate intrinsically labile complexes of highthermodynamic stability in the hope that such high thermodynamicstability will translate into a kinetic stability that is sufficient forin vivo applications. Indeed, little effort is currently being devotedto the preparation of nonlabile Gd(III) complexes that would in and ofthemselves enjoy a high kinetic stability. The problem seems to be quitesimply that such systems are hard to make. For instance, unlike thetransition metal cations which are bound well to porphyrins (asynthetically versatile ligand which is readily subject to modificationand which, at least for [Mn(III)TPPS]³ -, and other water solubleanalogues,³⁰⁻³⁴ shows good relaxivity and good tumor localizingproperties), Gd(III) forms only weak and/or hydrolytically unstablecomplexes with porphyrins,^(30c),34,40 although other simple macrocyclicamine- and imine-derived ligands³⁶,37,41 will support stable complexeswith certain members of the lanthanide series and do show some promise,as yet unrealized, of acting as supporting chelands for Gd(III)-basedMRI applications. It is a premise of the present invention thatnonlabile porphyrin-like Gd(III) complexes can be generated using an"expanded porphyrin" approach and that once made these complexes willprove to be useful contrast agents for MRI applications. In fact,texaphyrin is capable of stabilizing complexes with a variety of di- andtrivalent cations, including Cd²⁺, Hg²⁺, Y³⁺, and Nd³⁺. The observationthat a hydrolytically stable Nd³⁺ complex may be supported by texaphyrinbodes well for the use of texaphyrins in various gadolinium(III)-basedMRI applications. Unfortunately, as explained in greater detail inExample 4, all efforts to date to isolate a stable Gd³⁺ complex fromtexaphyrin in good yield in good yield have met with failure. It issuspected that this is because the complex is actually so water solublethat standard work-up methods fail. Consistent with this supposition isthe fact that fully characterized Sm³⁺, Eu³⁺, and Gd³⁺ (as well as Y³⁺)complexes have been prepared from the more hydrophobicdimethyl-texaphyrin. These complexes are obtained in roughly 25% yieldsfrom the corresponding reduced (methylene bridged) macrocyclic precursorusing the standard metal insertion and oxidation conditions discussedbelow and in Examples 1 and 2. Importantly, all of these complexes aresoluble in 1:1 methanol-water mixtures and all are quite stable undersuch potentially decomplexing conditions. The half-life of the Gd³⁺complex in 1:1 methanol-water at room temperature, for instance, is over5 weeks. Thus it is possible to use a texaphyrin-type approach togenerate hydrolytically stable gadolinium(III) complexes (something thatcannot be achieved using simple porphyrins.⁴⁰ Given this criticalresult, further modifications of the texaphyrin skeleton which shouldallow preparation of stable Gd³⁺ complexes with improved watersolubility or better biodistribution properties. In addition, by usingsuitable anionic side-chains, it should be possible to prepare neutralcomplexes with no net overall charge.

Such complexes would display a lower osmolality in aqueous solution.This would reduce the pain associated with their administration and havepositive clinical consequences. Thus the texaphyrin approach to MRI useof contrast agent development looks promising.

Literature citations in the following list are incorporated by referenceherein for the reasons cited.

REFERENCES

1. For an historical overview see: Budinger, T. F.; Lauterbur, P. C.Science 1984, 226, 288.

2. Morris, P. G. Nuclear Magnetic Resonance Imaging in Medicine andBiology, Claredon Press: Oxford; 1986.

3. For a review of biological applications of NMR see: MacKenzie, N. E.;Gooley, P. R. Med. Res. Rev. 1988, 8, 57.

4. For an introductory discussion of MRI contrast agents see: Tweedle,M. F.; Brittain, H. G.; Eckelman, W. C.; Gaughan, G. T.; Hagan, J. J.;Wedeking, P. W.; Runge, V. M. in Magnetic Resonance Imaging, 2nd ed.,Parrain, C. L., et al. Eds.; W. B. Saunders: Philadelphia; 1988, Vol. I,pp. 793-809.

5. For a comprehensive review of paramagnetic MRI contrast agents see:Lauffer, R. B. Chem. Rev. 1987, 87, 901.

6. Bloch, F. Phys. Rev. 1946, 70, 460.

7. (a) Bloembergen, N.; Purcell, E. M.; Pound, E. V. Phys. Rev. 1948,73, 679. (b) Solomon, I. Phys. Rev. 1955, 99, 559.

8. (a) Koenig, S. H.; Brown, R. D. III Magn. Res. Med. 1984, I, 437. (b)Koenig, S. H.; Brown, R. D. III Magn. Res. Med. 1984, 1, 478. (c)Koenig, S. H.; Brown, R. D. III Magn. Res. Med. 1985, 2, 159.

9. Tweedle, M. F.; Gaughan, G. T.; Hagan, J.; Wedeking, P. W.; Sibley,P.; Wilson, L. J.; Lee, D. W. Nucl. Med. Biol. 1988, 15, 31.

10. Burton, D. R.; Forsen S.; Karlstrom, G.; Dwek, R. A. Prog. NMRSprectr. 1979, 13, 1.

11. Carr, F. H.; Brown, J.; Bydder, G. M.; et al. Lancet 1984, 1, 484.

12. (a) Weinmann, H.-J.; Brasch, R. C.; Press, W. R.; Wesby, G. Am. J.Roentg. 1984, 142, 619. (b) Brasch, R. C.; Weinmann, H.-J.; Wesbey, G.E. Am J. Roentg. 1984, 142, 625.

13. (a) Runge, V. M.; Schoerner, W.; Niendorf, H. P.; et al. Mag. Res.Imaging 1985, 3, 27. (b) Runge, V. M.; Price, A. C.; Alleng, James, A.E. Radiology, 1985, 157(P), 37.

14. Koenig, S. H.; Spiller, M.; Brown, R. D. III; Wolf, G. L. Invest.Radiology 1986, 21, 697.

15. Johnston, D. L.; Lieu, P.; Lauffer, R. B.; Newell, J. B.; Wedeen, V.J.; Rosen, B. R.; Brady, T. J.; Okada, R. D. J. Nucl. Med. 1987, 28,871.

16. Schmiedl, U.; Ogan, M.; Paajanen, H.; Marotti, M. Crooks, L. E.;Brito, A. C.; Brasch, R. C. Radiology 1987, 162, 205.

17. Kornguth, S. E.; Turski, P. A.; Perman, W. H.; Schultz, R.; Kalinke,T.; Reale, R.; Raybaud, F. J. Neurosug. 1987, 66, 898.

18. (a) Lauffer, R. B.; Brady, T. J. Magn. Reson. Imaging 1985, 3, 11.(b) Lauffer, R. B.; Brady, T. J.; Brown, R. D.; Baglin, C.; Koenig, S.H. Magn. Reson. Med. 1986, 3, 541.

19. Southwood-Jones, R. V.; Earl, W. L.; Newman, K. E.; Merbach, A. E.J. Chem. Phys. 1980, 73, 5909.

20. Martell, A. E.; Smith, R. M. Critical Stability Constants, Plenum:New York; 1974, Vol. 4.

21. Cacheris, W. P.; Nickle, S. K.; Sherry, A. D. Inorg. Chem. 1987, 26,958.

22. Desreaux, J. F.; Loncin, M. F.; Spirlet, M. R. Inorg. Chim. Acta1984, 94, 43.

23. Chu, S. C.; Pike, M. M., Fossel, E. T.; Smith, T. W.; Balschi, J.A.; Springer, C. S., Jr. J. Man. Reson. 1984, 56, 33.

24. (a) Spirlet, M.-R.; Rebizant, J.; Desreaux, J. F.; Loncin, M. F.Inorg. Chem. 1984, 23, 359. (b) Spirlet, M.-R.; Rebizant, J.; Loncin, M.F.; Desreux, J. F. Inorg. Chem. 1984, 23, 4278.

25. Loncin, M. F.; Desreaux, J. F.; Merciny, E.; Inorg. Chem. 1986, 25,2646.

26. (a) Chang, C. A.; Rowland, M. E. Inorg. Chem. 1983, 22, 3866. (b)Chang, C. A.; Ochaya, V. O. Inorg. Chem. 1986, 25, 355. (c) Chang, C.A.; Sekhar, V. C. Inorg. Chem. 1987, 26, 1981.

27. Geraldes, C. F. G. C.; Sherry, A. D.; Brown, R. D. III; Koenig, S.H.; Magn. Reson. Med. 1986, 3, 242.

28. Lauffer, R. B.; Greif, W. L.; Stark, D. D.; Vincent, A. C.; Saini,S.; Wedeen, V. J.; Brady, T. J. J. Comput. Assist. Tomogr. 1985, 9, 431.

29. Lauffer, R. B.; Vincent, A. C.; Padmanabhan, S.; Meade, T. J. J. Am.Chem. Soc. 1987, 109, 2216.

30. (a) Chen, C.; Cohen, J. S.; Myers, C. E.; Sohn, M. FEBS Lett. 1984,168, 70. (b) Patronas, N. J.; Cohen, J. S.; Knop, R. H.; Dwyer, A. J.;Colcher, D.; Lundy, J.; Mornex, F.; Hambright, P. Cancer Treat. Rep.1986, 70, 391. (c) Lyon, R. C.; Faustino, P. J.; Cohen, J. S.; Katz, A.;Mornex, F.; Colcher, D.; Baglin, C.; Koenig, S. H.; Hambright, P. Magn.Reson. Med. 1987, 4, 24. (d) Megnin, F.; Faustino, P. J.; Lyon, R. C.;Lelkes, P. I.; Cohen, J. S. Biochim. Biophys. Acta 1987, 929, 173.

31. Jackson, L. S.; Nelson, J. A.; Case, T. A.; Burnham, B. F. Invest.Radiology 1985, 20, 226.

32. Fiel, R. J.; Button, T. M.; Gilani, S.; et al. Magn. Reson. Imaging1987, 5, 149.

33. Koenig, S. H.; Brown, R. D. III; Spiller, M. Magn. Reson. Med. 1987,4, 252.

34. Hambright, P.; Adams, C.; Vernon, K. Inorg. Chem. 1988, 27, 1660.

35. Smith, P. H.; Raymond, K. N. Inorg. Chem. 1985, 24, 3469.

36. For examples of lanthanide cryptates see: (a) Gansow, O. A.; Kauser,A. R.; Triplett, K. M.; Weaver, M. J.; Yee, E. L. J. Am. Chem. Soc.1977, 99, 7087. (b) Yee, E. L.; Gansow, O. A.; Weaver, M. J. J. Am.Chem. Soc. 1980, 102, 2278. (c) Sabbatini, N.; Dellonte, S.; Ciano, M.;Bonazzi, A.; Balzani; V. Chem. Phys. Let. 1984, 107, 212. (d) Sabbatini,N.; Dellont, S.; Blasse, G. Chem. Phys. Lett. 1986, 129, 541. (e)Desreux, J. F.; Barthelemy, P. P. Nucl. Med. Biol. 1988, 15, 9.

37. For examples of lanthanide complexes stablized by conventionalSchiff base macrocycles see: (a) Backer-Dirks, J. D. J.; Gray, C. J.;Hart, F. A.; Hursthouse, M. B.; Schoop, B. C. J. Chem. Soc., Chem.Commun. 1979, 774. (b) De Cola, L.; Smailes, D. L.; Vallarino, L. M.Inorg. Chem. 1986, 25, 1729. (c) Sabbatini, N.; De Cola, L.; Vallarino,L. M.; Blasse, G. J. Phys. Chem. 1987, 91, 4681. (d) Abid, K. K.;Fenton, D. E.; Casellato, U.; Vigato, P.; Graziani, R. J. Chem. Soc.,Dalton Trans. 1984, 351. (e) Abid, K. K.; Fenton, D. E. Inorg. Chim.Acta 1984, 95, 119-125. (f) Sakamoto, M. Bull Chem. Soc. Jpn. 1987, 60,1546.

38. Ochai, E.-I Bioinorganic Chemistry, an Introduction, Allyn andBacon: Boston; 1977, p. 168(Fe) and p 436(Mn).

39. For reviews see: (a) Cytochrome P-450: Structure, Mechanism, andBiochemistry, Ortiz de Montellano, P. R., Ed.; Plenum: New York, 1986.(b) Groves, J. T. Adv. Inorg. Biochem. 1979, I, 119.

40. (a) Buchler, J. W. in The Porphyrins, Dolphin, D. Ed., AcademicPress: New York; 1978, Vol. 1, Chapter 10. (b) Srivastava, T. S.Bioinorg. Chem. 1978, 8, 61. (c) Horrocks, W. DeW., Jr. J. Am. Chem.Soc. 1978, 100, 4386.

41. (a) Forsberg, J. H. Coord. Chem. Rev. 1973, 10, 195. (b) Bunzli;.J.-C.; Wesner, D. Coord. Chem. Rev. 1984, 60, 191.

EXAMPLE 9 Antibody Conjugates

Radioisotopes have long played a central role in the detection andtreatment of neoplastic disorders. Considerable research thereforecontinues to be devoted to improving their efficacity in medicalapplications. One of the more promising approaches in doing so involvesattacking radioisotopes to tumor-directed monoclonal antibodies andtheir fragments. Such monoclonal antibodies and their fragments localizeselectively at tumors; radiolabeled antibodies could therefore serve as"magic bullets" and allow the direct transport of radioisotopes toneoplastic sites thus minimizing whole body exposure to radiation.¹Considerable research is now being carried out along these lines (seereferences 2-11 for general reviews). Much, but certainly not all, isfocusing on the use of bifunctional metal chelating agents. It is thisapproach to radioimmunodiagnostics (RID) and therapy (RIT) that is mostclosely related to the present invention.

Bifunctional metal chelating agents for use in antibody conjugate-basedtreatment and diagnostic applications must satisfy two criticalcriteria: They must be capable of binding the radioisotope of interestand of attachment to the targeted antibody. Thus, these bifunctionalchelating agents must 1) have functional groups suitable for conjugationto the antibody, 2) form covalent linkages that are stable in vivo andwhich do not destroy the immunological competence of the antibody, 3) berelatively nontoxic, and 4) bind and retain the radiometal of interestunder physiological conditions.¹¹,15 The last of these conditions isparticularly severe. In contrast to MRI imaging where smallconcentrations of decomplexed cation can perhaps be tolerated, thepotential damage arising from "free" radioisotopes, released from theconjugate, can be very serious. Thus, for radioimmunological work, thecondition of nonlability must be strictly enforced. On the other hand,only nanomole concentrations of isotopes, and hence ligand, aregenerally required for RID and RIT applications, so that the concernsassociated with intrinsic metal and/or free ligand toxicity areconsiderably relaxed.

Needless to say, the above conditions must be met for each and everyisotope considered for RIT and RID work. Thus, from the point of view ofligand design and synthesis, the problem becomes one of identifying anisotope of medical advantage, designing a suitable ligand and attachingit to the antibody of choice either before or after metal binding.Historically, there has been a trade-off between choosing an idealisotope and one that can be readily complexed with existing difunctionalconjugates.

For the purposes of imaging, an ideal isotope should be readilydetectable by available monitoring techniques and induce a minimalradiation-based toxic response. In practice these and other necessaryrequirements implicate the use of a γ-ray emitter in the 100 to 250 KeVrange, which possesses a short effective half-life (biological and/ornuclear), decays to stable products, and, of course, is readilyavailable under clinical conditions.²⁻⁴ To date, therefore, mostattention has focused on ¹³¹ I (t_(1/2) =193h), ¹²³ I(t_(1/2) =13h),^(99m) Tc(t_(1/2) =6.0 h), ⁶⁷ Ga(t_(1/2) =78h), and ¹¹¹ In(t_(1/2)=67.4h) which come closest to meeting these criteria.¹⁶ Each of theseenjoys advantages and disadvantages with respect to antibody labelingfor RID. ¹³¹ I and ¹²³ I, for instance, are easily conjugated toantibodies (and other proteins) via simple electrophilic aromaticsubstitution of tyrosine residues.¹⁷ As a result, these isotopes haveseen wide use in immunological-based applications (RIT as well as RID).Unfortunately, such methods of conjugation are not particularly robustunder physiological conditions (metabolism of ¹³¹ I or ¹²³ I labeledproteins, for instance, produces free radioactive iodide anion) and as aresult can lead to a fair concentration of radioactivity at sites otherthan those targeted by the antibody-derived "magic bullet".¹⁷ Thisproblem is further exacerbated by the fact that the half-lives of both¹³¹ I and ¹²³ I are relatively inconvenient for optimal use, being toolong and too short, respectively, and the fact that ¹³¹ I is also a βemitter.¹⁶ 99m Tc, ⁶⁷ Ga, and ¹¹¹ In all suffer from the disadvantagethat they cannot be bound directly to the antibody in a satisfactoryfashion and require the use of a bifunctional conjugate. The chemistryof such systems is furthest advanced in the case of ^(99m) Tc, and anumber of effective ligands, are now available for the purpose of 99mTcadministration.²,12,18 This particular radioisotope, however, suffersfrom the serious disadvantage of having a very short half-life whichmakes it technically very difficult to work with. Both ⁶⁷ Ga and ¹¹¹ Inhave longer half-lives. In addition, both possess desirable emissionenergies. Unfortunately, both are "hard" cations with high chargedensity in their most common trivalent forms. Applications of theseradioisotopes in RID therefore requires the use of ligands which arecapable of forming stable, nonlabile complexes with these cations underphysiological conditions. Although considerable effort has been devotedto the development of DTPA-like systems¹⁹ which would be suitable for¹¹¹ In³⁺ (and, perhaps, ⁶⁷ G³⁺) binding and antibody functionalization,in all cases the complexes formed are too labile for safe and effectiveclinical use.²⁰ Indeed, at the present time, no suitable ligands existfor either ¹¹¹ In³⁺ or ⁶⁷ Ga³⁺ which form stable nonlabile complexes andwhich might be suitable for radioimmunological applications. Asdescribed elsewhere herein texaphyrin forms a kinetically andhydrolytically stable complex with In³⁺. Such a ligand system could beelaborated and serve as the critical core of a bifunctional conjugatefor use in ¹¹¹ In-based RID.

Many of the same considerations hold true for radioisotope-based therapyas do for radioisotope-based diagnostics: An ideal isotope must also bereadily available under clinical conditions (i.e. from a simpledecay-based generator),²⁰ possess a reasonable half-life (i.e. on theorder of 6 hours to 4 weeks), and decay to stable products. In addition,the radioisotope must provide good ionizing radiation (i.e. in the 300KeV to 3 MeV range). In practice this means using either an α emitter ormedium to high energy β emitter.¹⁶ Although few α emitters are availablefor therapeutic use (²¹¹ At is an exception), a fair number of βemitters, including ¹³¹ I, are currently receiving attention as possiblecandidates for RIT. Among the more promising, are ¹⁸⁶ Re (t_(1/2) =90 h,⁶⁷ Cu (t_(1/2) =58.5 h), and ⁹⁰ Y (t_(1/2) =65 h). Of these, ⁹⁰ Y iscurrently considered the best,¹⁶,21 with an emission energy of 2.28 MeV,it is calculated to deliver roughly 3 to 4 times more energy (dose) tothe tumor per nanomole than either ¹⁸⁶ Re or ⁶⁷ Cu. Unfortunately, atthe present time, good immuno-compatible chelands exist for only ¹⁸⁶ Reand ⁶⁷ Cu: The former may be attached using the same ligands as weredeveloped for ^(99m) Tc,¹⁸ and the latter via the rationally-designedactivated porphyrins developed by Prof. Lavallee of Hunter College andthe Los Alamos INC-11 team.¹⁵ Although these new porphyrin-basedsystems, in particular, show real promise, being apparently far superletto the existing DTPA or DOTA-type systems,¹⁴ further benefits should bederived from a bifunctional conjugate which is capable of formingstable, nonlabile complexes with ⁹⁰ Y³⁺ (which cannot be done withporphyrins). The texephyrin ligand of the present invention not onlyforms stable complexes with In³⁺ but also binds Y³⁺ effectively. Atexaphyrin-type bifunctional conjugate should be developed for use in¹¹¹ In-based RID and could also find important application in ⁹⁰ Y-basedRIT. This application outlines ways in which such putative bifunctionalconjugates may be prepared.

The observation that complexes of both Y³⁺ and In³⁺ may be preparedaugurs well for the use of texaphyrin-type systems as conjugates inimmunological applications: Both ⁹⁰ Y and ¹¹¹ In could conceivably beattached to an antibody of choice using a functionalized texaphyrin. Inthis regard it is important to note that both the Y³⁺ and In³⁺ complexesof texaphyrin are formed rapidly (insertion and oxidation times are lessthan 3 hours) from the methylene-linked reduced precursor, and arehydrolytically stable in 1:1 methanol-water mixtures (the half-lives fordecomplexation and/or ligand decomposition exceed 3 weeks in both cases.

A further advantage of having developed or developing a wide variety ofsolubilized texaphyrins, such as those shown in FIG. 27A, FIG. 27B, FIG.27C, FIG. 27D, FIG. 31A, FIG. 31B and FIG. 31C, is that many of thesewould be suitable for further functionalization. For instance, treatmentof texaphyrins 7_(B) or 26_(D) with thionyl chloride or p-nitrophenolacetate would generate activated acyl species suitable for attachment tomonoclonal antibodies or other biomolecules of interest. Alternatively,standard in situ coupling methods (e.g. 1,1'-carbonyldiimidazole(CDI)^(26a)) could be used to effect the same sort of conjugation. Ineither case, the ability to attach and deliver a potent photosensitizerdirectly to a tumor locus could have tremendous potential benefit in thetreatment of neoplastic disorders. In addition, it is precisely thisapproach which will allow a variety of useful radioisotopes such as ⁹⁰ Yand ¹¹¹ In to be attached to a monoclonal antibody. This could prove tobe of immense benefit in the development of this important approach totumor detection and treatment.

Literature citations in the following list are incorporated by referenceherein for the reasons cited.

REFERENCES

1. Pressman, D.; Korngold, L. Cancer 1953, 6, 619.

2. Clinical Nuclear Medicine, Matin, P., Ed., Medical Examination: NewYork; 1981.

3. Radioimmunoimaging and Radioimmunotherapy, Burchiel, S. W. andRhodes, B. A., Eds., Elsevier: New York; 1983.

4. Nuclear Imaging in Oncology, Kim, E. E.; Haynie, T. P., Eds.,Appleton-Century-Crofts: Norwalk, Conn.; 1984.

5. Chevru, L. R.; Nunn, A. D.; Loberg, M. D. Semin. Nucl. Med. 1984, 12,5.

6. Order, S. E. Compr. Therapy 1984, 10, 9.

7. Spencer, R. P. Nuclear Medicine, Medical Examination: New York; 1984.

8. Radiopharmaceuticals and Labelled Compounds 1984 (proceedings of a1984 conference of the same name), International Atomic Energy Agency:Vienna, 1985.

9. DeLand, F. H.; Goldenberg, D. M. Semin. Nucl. Med. 1985, 15, 2.

10. Radiopharmaceuticals: Progress and Clinical Perspectives, Fritzberg,A. R., Ed., CRC Press: Boca Raton, Fla.; 1986.

11. Goldenberg, D. M.; Goldenberg, H.; Primus, F. J. inImmunoconjugates: Antibody Conjugates in Radioimaging and Therapy ofCancer, Vogel, C.-W., Ed., Oxford University Press: Oxford; 1987, pp.259-280.

12. Eckelman, W. C.; Paik, C. H.; Reba, R. C. Cancer Res. 1980, 40,3036.

13. Cole, W. C.; DeNardo, S. J.; Meares, C. F.; McCall, M. J.; DeNardo,G. L.; Epstein, A. L.; O'brien, H. A.; Moi, M. K. J. Nucl. Med. 1987,28, 83.

14. Deshpande, S. V.; DeNardo, S. J.; Meares, C. F.; McCall, M. J.;Adams, G. P.; Moi, M. K.; DeNardo, G. L. J. Nucl. Med. 1988, 29, 217.

15. Mercer-Smith, J. A.; Roberts, J. C.; Figard, S. D.; Lavallee, D. K.in Antibody-Mediated Delivery Systems, Rodwell, J. D. Ed., MarcelDekker: New York; 1988, pp. 317-352.

16. (a) O'Brien, H. A., Jr. in reference 119, pp. 161-169. (b) Wessels,B. W.; Rogus, R. D. Med. Phys. 1984, 11, 638. (c) Jungerman, J. A.; Yu,K.-H. P.; Zanelli, C. I. Int. J. Appl. Radiat. Isot. 1984, 9, 883. (d)Humm, J. L. J. Nucl. Med. 1986, 27, 1490.

17. See for instance: (a) Primus, F. J.; DeLand, F. H.; Goldenberg, D.M. in Monoclonal Antibodies and Cancer, "Wright, G. L. Ed., MarcelDekker: New York; 1984, pp. 305-323. (b) Weinstein, J. N.; Black, C. D.V.; Keenan, A. M.; Holten, O. D., III; Larson, S. M.; Sieber, S. M.;Covell, D. G.; Carrasquillo, J.; Barbet, J.; Parker, R. J. in"Monoclonal Antibodies and Cancer Therapy," Reisfeld, R. A. and Sell,S., Eds., Alan R. Liss: New York; 1985, pp. 473-488.

18. Burns, H. D.; Worley, P.; Wagner, H. N., Jr.; Marzilli, L.; Risch,V. in The Chemistry of Radiopharmaceuticals, Heindel, N. D.; Burns, H.D.; Honda, T.; Brady, L. W., Eds., Masson: New York; 1978.

19. Paik, C. H., Ebbert, M. A.; Murphy, P. R.; Lassman, C. R.; Reba, R.C.; Eckelman, W. C.; Pak, K. Y.; Powe, J.; Steplewski, Z.; Koprowski, H.J. Nucl. Med. 1983, 24, 1158.

20. See for instance: Hnatowich, D. J.; Childs, R. L.; Lanteigne, D.;Najafi, A. J. Immunol. Meth. 1983, 65, 147.

21. See for instance: Hnatowich, D. J.; Virzi, F.; Doherty, P. W. J.Nucl. Med. 1985, 26, 503.

22. Katagi, T.; Yamamura, T.; Saito, T.; Sasaki, Y. Chem. Lett. 1981,503.

23. Sessler, J. L.; Johnson, M. R.; Lynch, V. J. Org. Chem. 1987, 52,4394.

24. Niclas, H. J.; Bohle, M.; Rick, J.-D.; Zeuner, F.; Zolch, L. Z.Chem. 1985, 25, 137.

25. Beilstein 4th ed., Band 14, p. 785.

26. (a) Paul, R.; Anderson, G. W. J. Am. Chem. Soc. 1960, 82, 4596. (b)Davis, M.-T. B.; Preston, J. F. Anal. Biochem. 1981, 116, 402. (c)Anderson, G. W.; Zimmerman, J. E; Callahan, F. M. J. Am. Chem. Soc.1964, 86, 1839.

27. Vollhardt, K. P. C. Synthesis 1985, 765.

28. Kati, H. A.; Siddappa, S. Indian J. Chem. 1983, 22B, 1205.

29. Hove, E.; Horrocks, W. D. J. Am. Chem. Soc. 1978, 100, 4386.

30. Furhop, J.-H.; Smith, K. M. in Porphyrins and Metalloporphyrins,Smith, K. M., Ed., Elsevier: Amsterdam; 1975.

What is claimed is:
 1. A compound having the structure: ##STR16##wherein R is H or CH₃.
 2. A method of assessing biodistribution of aphotosensitive texaphyrin comprising the step of monitoring fluorescenceof the texaphyrin wherein fluorescence indicates the presence oftexaphyrin.
 3. The method of claim 2 wherein the texaphyrin is atexaphyrin-diamagnetic metal complex.
 4. The method of claim 2 whereinthe step of monitoring fluorescence is at a wavelength about 20-35 nmlonger than an absorbing wavelength.
 5. The method of claim 2 whereinthe biodistribution is assessed in blood, human serum or neoplastictissue.
 6. The method of claim 2 wherein the texaphyrin has thestructure: ##STR17## wherein M is H or a diamagnetic metal cation;R₁ isalkyl, carboxy or carboxyalkyl; R₂ and R₃ are independently hydrogen,halide other than iodide, nitro, sulfonyl, cyano, alkyl, alkoxy,alkoxyalkyl, polyether, carboxy or carboxyalkyl; or R₂ and R₃ togetherform an alkenylene bridge of the formula --CH═CH--CH═CH-- optionallysubstituted with sulfonyl; the molecular weight of any one of R₁, R₂, orR₃ is less than or equal to about 1000 daltons; and n is an integer lessthan or equal to
 2. PG,146
 7. The method of claim 6 wherein R₁ is CH₂CH₃.
 8. The method of claim 6 wherein each of R₂ and R₃ is methyl; or R₂is hydrogen and R₃ is selected from the group consisting of methyl,methoxy, halide other than iodide, nitro, sulfonyl, --COOH, and --O(CH₂CH₂ O)₂ CH₃ ; or R₂ and R₃ together are an alkenylene bridge of theformula --CH═CH--CH═CH-- or --C(SO₃ --)═CH--CH═CH--.
 9. A method ofdetecting the presence of texaphyrin in solution or in tissuecomprising:irradiating the solution or tissue; and monitoringfluorescence emitted from the irradiated solution or tissue; whereinfluorescence indicates the presence of texaphyrin.
 10. The method ofclaim 9 wherein the texaphyrin is a texaphyrin-diamagnetic metalcomplex.
 11. The method of claim 9 wherein the irradiating is at awavelength range of about 700-800 nm and the emitted fluorescence ismonitored at a wavelength range at about 5-40 nm longer than theirradiating wavelength.
 12. The method of claim 9 wherein the solutionor tissue is blood, human serum or neoplastic tissue.
 13. The method ofclaim 9 wherein the solution or tissue contains a virus.
 14. The methodof claim 13 wherein the virus is an enveloped virus.
 15. The method ofclaim 9 wherein the texaphyrin has the structure: ##STR18## wherein M isH or a diamagnetic metal cation;R₁ is alkyl, carboxy or carboxyalkyl; R₂and R₃ are independently hydrogen, halide other than iodide, nitro,sulfonyl, cyano, alkyl, alkoxy, alkoxyalkyl, polyether, carboxy orcarboxyalkyl; or R₂ and R₃ together form an alkenylene bridge of theformula --CH═CH--CH═CH-- optionally substituted with sulfonyl; themolecular weight of any one of R₁, R₂, or R₃ is less than or equal toabout 1000 daltons; and n is an integer less than or equal to
 2. 16. Themethod of claim 15 wherein R₁ is CH₂ CH₃.
 17. The method of claim 15wherein each of R₂ and R₃ is methyl; or R₂ is hydrogen and R₃ isselected from the group consisting of methyl, methoxy, halide other thaniodide, nitro, sulfonyl, --COOH, and --O(CH₂ CH₂ O)₂ CH₃ ; or R₂ and R₃together are an alkenylene bridge of the formula --CH═CH--CH═CH-- or--C(SO₃ --)═CH--CH═CH--.